Glycogen biosynthetic enzymes in plants

ABSTRACT

The present invention is directed to the modification of reserve polysaccharides in plants. Specifically, it has been found that host plants can be successfully transformed with a nucleic acid sequence capable of expressing a chimeric reserve polysaccharide modification enzyme gene sequence which will synthesize novel reserve polysaccharides in plants or convert the transformed plant&#39;s endogenous starch reserves to novel starch degradation products.

RELATED APPLICATION DATA

The present application is a continuation application of U.S. patentapplication Ser. No. 08/016,881, filed on Feb. 11, 1993, which is acontinuation-in-part of U.S. patent application Ser. No. 07/735,065,filed on Jul. 24, 1991, now U.S. Pat. No. 5,349,123, which in turn is acontinuation-in-part of abandoned U.S. patent application Ser. Nos.07/632,383 filed on Dec. 21, 1990 and 07/731,226 filed on Jul. 16, 1991;and a CIP of 07/536,392 filed on Jun. 11, 1990, now abandoned.

TECHNICAL FIELD

This invention relates to transgenic plants and, more particularly, tomethods and compositions which modify the biosynthesis and degradationpathways of reserve polysaccharides in plants.

BACKGROUND OF THE INVENTION

In the animal kingdom, nonvascular plants, fungi, yeast and bacteria,the primary reserve polysaccharide is glycogen. Glycogen is a D-glucosepolysaccharide containing linear molecules with α-1,4 glycosyl linkagesand is branched via α-1,6 glycosyl linkages. Although glycogen isanalogous to starch from a linkage comparison, glycogen exhibits adifferent chain length and degree of polymerization. In bacteria, forexample, the α-1,6 glycosyl linkages constitute only approximately 10%of the total linkages, indicating that the majority of the glycogenpolymer resides as linear glucose units.

In vascular plants, reserve polysaccharides are stored in roots, tubersand seeds in the form of starch. Starch, a complex polymer of D-glucose,consists of a mixture of linear chain (amylose) and branched chain(amylopectin) glucans. Starches isolated from different plants are foundto have distinct proportions of amylose. Typically, amylose comprisesfrom about 10-25% of plant starch, the remainder being the branchedpolymer amylopectin. Amylopectin contains low molecular weight chainsand high molecular weight chains, with the low molecular weight chainsranging from 5-30 glucose units and the high molecular weight chainsfrom 30-100 or more. The ratio of amylose/amylopectin and thedistribution of low molecular weight to high molecular weight chains inthe amylopectin fraction are known to affect the properties, such asthermal stabilization, retrogradation, and viscosity, and therefore theutility of starch. The highest published low m.w./high m.w. chain ratios(on a weight basis) in amylopectin are 3.9/1 for waxy corn starch, whichhas unique properties. Additionally, duwx, which has slightly morebranch points than waxy, also has further unique properties.

In addition, starches from different plants or plant parts often havedifferent properties. For example, potato starch has differentproperties than other starches, some of which may be due to the presenceof phosphate groups. In some plant species, mutants have been identifiedwhich have altered contents of amylose and amylopectin. Mutations thataffect the activity of starch-branching enzyme in peas, for example,result in seeds having less starch and a lower proportion ofamylopectin. Also, mutations in the waxy locus of maize, which encodes astarch granule bound starch synthase, result in plants which produceamylopectin exclusively. Similarly, a potato mutant has been identifiedwhose. starch is amylose-free (Hovenkamp-Hermelink et al. Theor. Appl.Genet. (1987) 75:217-221). It has been found that varying the degree ofstarch branching can confer desirable physical properties; other changesin the characteristics of native starch could result in the productionof polymers with new applications.

Cyclodextrins are the products of enzymatic starch degradation by aclass of amylases termed cyclodextrin glycosyltransferase (CGT) enzymes.The family of cyclodextrins contains three major and several minorcyclic oligosaccharides which are composed of a number of homogenouscyclic α-1,4-linked glucopyranose units. The cyclodextrin having sixglucopyranose units is termed α-cyclodextrin (also know as Schardinger'sα-dextrin, cyclomaltohexaose, cyclohexaglucan, cyclohexaamylose, α-CD,ACD and C6A). The seven unit cyclodextrin is termed β-cyclodextrin (alsoknown as Schardinger's β-dextrin, cyclomaltoheptaose, cycloheptaglucan,β-CD, BCD and C7A). The eight unit cyclodextrin is termed y-cyclodextrin(also known as Schardinger's y-dextrin, cyclomaltooctaose,cyclooctaglucan, cyclooctaamylose, γ-CD, GCD and C8A).

The cyclic nature of cyclodextrins allows them to function as clathrates(inclusion complexes) in which a guest molecule is enclosed in thehydrophobic cavity of the cyclodextrin host without resort to primaryvalence forces. Thus, the components are bound as a consequence ofgeometric factors, and the presence of one component does notsignificantly affect the structure of the other component. Complexing ahydrophobic compound with cyclodextrin increases the stability andsolubility of the hydrophobic compound. Applications of this phenomenahave been found in many fields including pharmaceuticals, foodscosmetics and pesticides.

In pharmaceutical applications, complexing a drug with cyclodextrins fororal delivery can have many advantages. Among the benefits are thetransformation of liquids into solids which can be formed into tablets,stabilization of drugs against volatilization and oxidation, reductionof bad taste or smell, improvement in the rate of dissolution of poorlysoluble drugs and increases in blood levels of poorly water solubledrugs (Pitha, in Controlled Drug Delivery, Bruck, ed. Vol. 1, p. 125,(1983) CRC Press). From the limited research done on parenteraladministration of cyclodextrin-complexed drugs, some of the sameadvantages found for oral delivery can also be observed. The undesirableside effects of drugs can be reduced with complexation withcyclodextrins. Such side affects include gastric irritation from oraldelivery, local irritation and hemorrhagic areas from intramuscularinjection, and local irritation from eye-drops (Szejtli, J.,Cyclodextrin Technology, Kluwer Academic Publications, Boston (1988),pp. 186-306).

The addition of cyclodextrins to food products or cosmetics can alsohave many effects. In spices, food flavoring or perfume fragrances,cyclodextrins protect against oxidation, volatility, and degradation byheat or light (Hashimoto, H., "Application of Cyclodextrins to Food,Toiletries and Other Products in Japan," in Proceedings of the FourthInternational Symposium of Cyclodextrins, O. Huber and J. Szejtli, eds.(1988) pp. 533-543). Cyclodextrins can also eliminate or reduceundesirable smells or tastes, and modify food or cosmetic textures.

Complexing pesticides with cyclodextrins can increase thebioavailability of poorly wettable or slightly soluble substances, andtransform volatile liquids or sublimable solids into stable solidpowders (Szejtli, J. (1988) supra at pp. 335-364; U.S. Pat. No.4,923,853). Pesticides which are sensitive to light, heat or oxygendegradation can be stabilized by complexing with cyclodextrins.

Currently, production of cyclodextrins begins with the cultivation of anappropriate microorganism, e.g., Bacillus macerans, and separation,purification and concentration of the amylase enzyme. The enzyme is thenused to convert a starch substrate to a mixture of cyclic and acyclicdextrins. Subsequent separation and purification of cyclodextrins isthen required. The bacterial strain from which the enzyme is isolatedand the length of time the starch conversion is allowed to progressdetermines the predominant form of cyclodextrin produced. Manufacturesof α-cyclodextrins attempt to manipulate the reaction to preferentiallymake the specific cyclodextrin, however, the process is not easilycontrolled, and a mixture of cyclodextrins is obtained. At the presenttime β-cyclodextrin is the most widely commercialized form ofcyclodextrin because the β-form is much cheaper to produce than the α-or γ-cyclodextrins.

In 1987, the U.S. market for cyclodextrins was predicted to reach $50million per year within 2 years; that figure would double if the U.S.Food and Drug Administration approved the use of cyclodextrins in food(Seltzer, R., Chem. Eng. News, (May 1987) pp. 24-25). The world marketis estimated to be twice the U.S. figure (Szejtli, J. (1988) supra at p.viii). The potential U.S. market for cyclodextrins has been predicted toreach as high as $245 million per year (Anon., Bioproc. Technol.,November 1987). There is potentially a large market waiting to be tappedif the cost of cyclodextrins could be lowered through alternativeproduction methods.

With the development of genetic engineering techniques, it is nowpossible to transfer genes from a variety of organism into the genome ofa large number of different plant species. This process is preferable toplant breeding techniques whereby genes can only be transferred from oneplant in a species to another plant in the same or a closely relatedspecies. It would thus be desirable to develop plant varieties throughgenetic engineering, which have increased capacity for starch synthesis,altered amylose/amylopectin ratios, altered distribution of low to highmolecular weight chains in the amylopectin fraction and also starcheswith novel molecular weight characteristics. In this manner, usefulstarches with a variety of viscosity or texture differences may beobtained.

In addition, recognizing the disadvantages of bacterial-derivedCGT-mediated cyclodextrin production, it is considered desirable toproduce cyclodextrins where CGT is the expression product of arecombinant gene transferred into a plant host. In this method,generically known as molecular farming, plants are transformed with astructural gene of interest and the product extracted and purified froma harvested field of the transgenic plants. For example, human serumalbumin has been produced in transgenic tobacco and potato (Sijmons, P.C. et al., Bio/Technology (1990) 8:217-221).

Extending the idea of molecular farming to cyclodextrins provides ameans to lower production costs. One particularly desirable host plantfor such transformation is potato because of the large amount of starchproduction in potato tubers. A typical tuber contains approximately 16%of its fresh weight as starch (Burton, W. G., The Potato (1966) 3rdEdition, Longman Scientific and Technical Publications, England, p.361). Transformation of potato plants with the bacterial CGT structuralgene linked to a tuber-specific promoter and a leader directing theenzyme, for example, to the amyloplast, provides a means to producelarge quantities of cyclodextrins in tubers.

To this end, nucleic acid sequences which encode glycogen biosyntheticor degradative enzymes are desirable for study and manipulation of thestarch biosynthetic pathway. In particular, these enzymes may beexpressed in plant cells using plant genetic engineering techniques andtargeted to a plastid where starch synthesis occurs. It was thereforeconsidered desirable to apply recombinant deoxyribonucleic acid (rDNA)and related technologies to provide for modified reserve polysaccharidesin transgenic plants.

Proceeding from the seminal work of Cohen & Boyer, U.S. Pat. No.4,237,224, rDNA technology has become available to provide novel DNAsequences and to produce heterologous proteins in transformed cellcultures. In general, the joining of DNA from different organisms relieson the excision of DNA sequences using restriction endonucleases. Theseenzymes are used to cut donor DNA at very specific locations, resultingin gene fragments which contain the DNA sequences of interest.Alternatively, structural genes coding for desired peptides andregulatory control sequences of interest can now be producedsynthetically to form such DNA fragments.

These DNA fragments usually contain short single-stranded tails at eachend, termed "sticky-ends". These sticky-ended fragments can then beligated to complementary fragments in expression vehicles which havebeen prepared, e.g., by digestion with the same restrictionendonucleases. Having created an expression vector which contains thestructural gene of interest in proper orientation with the controlelements, one can use this vector to transform host cells and expressthe desired gene product with the cellular machinery available.Recombinant DNA technology provides the opportunity for modifying plantsto allow the expression of desirable enzymes in planta.

However, while the general methods are easy to summarize, theconstruction of an expression vector containing a desired structuralgene is a difficult process and the successful expression of the desiredgene product in significant amounts while retaining its biologicalactivity is not readily predictable. Frequently, bacterial-derived geneproducts are not biologically active when expressed in plant systems.

To successfully modify plants using rDNA, one must usually modify thenaturally occurring plant cell in a manner in which the cell can be usedto generate a plant which retains the modification. Even in successfulcases, it is often essential that the modification be subject toregulation. That is, it is desirable that the particular gene beregulated as to the differentiation of the cells and maturation of theplant tissue. In the case of glycogen synthase, ADP-glucosepyrophosphorylase and/or cyclodextrin glycosyltransferase, it is alsoimportant that the modification be performed at a site where the productwill be directed to contact the reserve polysaccharide regions of themodified plant. Thus, genetic engineering of plants with rDNA presentssubstantially increased degrees of difficulty.

In addition, the need to regenerate plants from the modified cellsgreatly extends the period of time before one can establish the utilityof the genetic construct. It is also important to establish that theparticular constructs will be useful in a variety of different plantspecies. Furthermore, one may wish to localize the expression of theparticular construct in specific sites and it is desirable that thegenetically modified plant retain the modification through a number ofgenerations.

RELEVANT LITERATURE

The structural genes encoding the E. coli glycogen biosynthetic enzymeshave been cloned (Okita, et al. (1981) J. Biol. Chem. 256: 6944-6952)and their nucleic acid sequences determined (Preiss, J. (1984) Ann. Rev;Microbiol. 38:419-458; Kumar et al. (1986) J. Biol. Chem.261:16256-16259). Genes encoding mammalian glycogen synthases have alsobeen cloned and their nucleic acid sequences determined (Browner, et al.Proc. Nat. Acad. Sci. (1989) 86:1443-1447; Bai, et al., J. Biol Chem.(1990) 265:7843-7848).

SUMMARY OF THE INVENTION

By this invention, nucleic acid constructs comprising at least onechimeric reserve polysaccharide modification enzyme gene sequence andpromoter and control sequences operable in plant cells, are provided.

In particular, one aspect of this invention relates to constructscomprising sequences relating to reserve polysaccharide biosyntheticenzymes, such as glycogen biosynthetic enzymes, glycogen synthase and/orADP-glucose pyrophosphorylase. Another aspect of the invention relatesto constructs comprising sequences relating to polysaccharidedegradation enzymes, including amylases such as cyclodextringlycosyltransferases.

In one aspect of the invention, a sequence encoding a desired enzyme isjoined to a sequence which encodes a transit peptide that provides fortranslocation of the enzyme to a plastid.

Other constructs of this invention provide sequences for transcriptionof the selected enzyme sequences in plant cells. To this end,transcriptional initiation regions that function to regulate expressionof genes in plants are considered. Of particular interest are thoseregulatory regions that preferentially direct expression of genes inroots, tubers, and seeds, or in other plant parts that synthesizereserve starch. In addition, constructs may contain sequences encoding amarker enzyme for selection of transformed cells.

Expression constructs which comprise sequences which provide fortranscriptional and translational regulation in plant cells of thesequences encoding the desired enzymes are of special interest. Theseconstructs include, in the 5'-3' direction of transcription, atranscriptional/translational initiation control region, a sequenceencoding a selected enzyme in reading frame, and atranscription/translation termination region, wherein the sequenceencoding the enzyme is under the regulatory control of the initiationand termination regions. Expression constructs may also containsequences which encode a transit peptide that provides for translocationof the enzymes to plastids and/or a marker enzyme.

Another aspect of the invention involves vectors which comprisesequences providing for transfer of desired sequences and integrationinto the genome of a plant cell. For example plant transformationvectors may include Agrobacterium T-DNA border region(s) to provide fortransfer of the sequences to the plant cell.

Also considered part of this invention are plant cells containingnucleic acid sequences of the desired enzyme. Such plant cells areobtainable through transformation techniques which utilize, e.g.,Agrobacterium to transfer DNA to the plant cells or through directtransfer techniques such as DNA bombardment, electroporation ormicroinjection. Plant cells containing the desired sequences can beregenerated to yield whole plants containing the sequences.

In yet another aspect of this invention, plant cells containing thedesired enzymes or having reduced or increased starch precursor enzymesare considered. Of particular interest are plant cells in starch storageorgans, such as roots, tubers or seeds. It is preferable that the enzymebe located in plastids, where starch synthesis occurs, and morepreferably in amyloplasts, where reserve starch is synthesized andstored.

Further, it can be recognized that the modulation of polysaccharidemodification enzymes in these plant cells has implications for modifyingthe starch content and/or composition of these cells. In this manner,plants or plant parts which synthesize and store starch may be obtainedwhich have increased or decreased starch content and modified starchrelated properties such as specific gravity, free sugar content and/ornovel and useful starches. In particular, potato starch having decreasedamylose and modified amylopectin may be produced and furtherapplications to modify starches consisting entirely of amylopectin suchas that of waxy maize or a mutant potato, are also considered.Similarly, the starch from these plant parts can be harvested for use incommercial applications, or can be modified in planta to produce desiredstarch degradation products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depicts a DNA sequence (SEQ ID NO: 11) for the E. coilglycogen synthase gene, glgA, generated, through Polymerase ChainReaction (PCR) from E. coli K-12 618;

FIGS. 2A-2E depict the translated amino acid sequence (SEQ ID NO:12) ofthe PCR generated glgA gene;

FIGS. 3A-3E depict DNA sequence (SEQ ID NO: 13) and the translated aminoacid sequence (SEQ ID NO:14) of the PCR generated E. coli ADP-glucosepyrophosphorylase gene, glgC, from E. coli K-12 618;

FIGS. 4A-4B depict the DNA sequence which encodes a SSU transit peptidefrom soybean plus 48 bp of DNA which encodes a mature SSU protein frompea, together with the amino acid sequence encoded by the reading frame(upper sequence); The DNA sequence of FIG. 4 and the translated aminoacid sequences in three reading frames are represented as (SEQ. ID NO:15-20);

FIGS. 5A-5C depict a comparison of DNA sequences from patatin 5'untranslated regions from Solanum tuberosum varieties Kennebec (topsequence, SEQ ID NO: 21) (generated by PCR) and Maris Piper (bottomsequence, SEQ ID NO: 22);

FIGS. 6A-6C depict a comparison of DNA sequences from patatin 5'untranslated regions from Solanum tuberosum varieties Russet Burbank(top sequence, SEQ ID NO: 22) (generated by PCR) and Maris Piper (bottomsequence, SEQ ID NO: 24);

FIGS. 7A-7I depicts a comparison of DNA sequences for native Klebsiellapneumoneae cyclodextrin glycosyltransferase (bottom sequence, SEQ ID NO:25) and PCR-generated pCGT2 cyclodextrin glycosyltransferase (topsequence, SEQ ID NO: 26) (absence of bar between bases indicatesdifference in the two sequences); and

FIGS. 8A-8C depicts a comparison of amino acid sequences for nativeKlebsiella pneumoneae cyclodextrin glycosyltransferase (bottom sequence,SEQ ID NO: 27) and pCGT2 cyclodextrin glycosyltransferase (top sequence,SEQ ID NO: 28) (absence of bar between residues indicates difference inthe two sequences).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the expression of novel reservepolysaccharide modification enzyme gene sequences in plants. Inparticular, this invention is directed to a plant cell having nucleicacid sequences encoding such enzymes integrated in its genome as theresult of genetic engineering. Cells containing a DNA or RNA (mRNA)sequence encoding the enzyme, as well as cells containing the enzyme,are also provided. Plants and, more particularly, plant parts may alsobe obtained which contain such enzyme gene sequences and/or suchenzymes.

In considering the present reserve polysaccharide modification enzymes,there are two major classes presented: Biosynthetic enzymes whichproduce novel reserve polysaccharides, and starch degradation enzymeswhich produce novel starch degradation products. Representative of thefirst class of such enzymes include glycogen biosynthetic enzymes, whichare not known to be endogenous to vascular plants.

The biosynthetic steps involved in glycogen synthesis in E. coliinclude: 1) the formation of ADP-glucose from ATP and glucose1-phosphate, 2) the transfer of a glucose unit from ADP-glucose to apreformed maltodextrin primer via an α-1,4 linkage, and 3) the formationof α-1,6 glucosyl linkages from glycogen. The bacterial enzymes whichcatalyze the above reactions are ADP-glucose pyrophosphorylase (EC2.7.7.27), glycogen synthase (EC 2.4.1.21), and Q-enzyme or branchingenzyme (EC 2.4.1.18), respectively. The genes encoding these enzymeshave been cloned and are also known as glgC, glgA, and glgB,respectively.

The pathway of glycogen biosynthesis in mammals is similar to that inbacteria, an exception being that UDP-glucose is the preferred glucosedonor. The mammalian enzymes which catalyze glycogen biosyntheticreactions similar to those in bacteria are glucose-1-phosphateuridylyltransferase, glycogen synthase (EC 2.4.1.11), and 1,4-α-glucanbranching enzyme. Genes encoding human muscle and rat liver glycogensynthases have been cloned and their sequences determined.

In particular, the glycogen biosynthesis enzyme glycogen synthase (glgA)is of special interest. The E. coli glycogen synthase is of particularinterest in that the enzyme is similar to plant starch synthase withrespect to being non-responsive to allosteric effectors or chemicalmodifications. Expression of a glycogen synthase enzyme in a plant hostdemonstrates biological activity even within an intact plant cell.Namely, potato plants having glgA expressed in potato tubers result intubers having a deceased specific gravity; specific gravity being acommonly used measurement with respect to dry matter and starch contentsof potato tubers (W. G. Burton, in The Potato, Third Edition, pub.Longman Scientific & Technical (1989) Appendix II, pp. 599-601). Furtheranalysis of transgenic tubers having decreased specific gravityindicates that the starch in these tubers is modified. In particular,the percentage of amylose is decreased and the ratio of low m.w./highm.w. chains in the amylopectin fraction is increased. This phenotypiceffect in planta is indicative of glgA biological activity. Additionaldisclosure concerning glycogen biosynthetic enzymes can be found in U.S.patent application Ser. No. 07/735,065, filed on Jul. 16, 1991 and U.S.patent application Ser. No. 07/632,383, filed on Dec. 21, 1990, nowabandoned, the complete specifications of which are incorporated hereinby this reference.

Other phenotypic starch modifications resulting from biological activityof glycogen biosynthetic enzymes in plants are also considered in thisinvention. Such altered phenotypes may result from enzymatic activity ofthese proteins on plant starch precursors, or from the inhibition ofplant starch biosynthetic enzyme activities. Inhibition of plantenzymes, for example, could result through the production of inactiveforms of the plant enzymes as the result of association with theglycogen biosynthetic enzymes. The inhibition of plant enzymes may thenlead to plants having altered starch (such as branching patterns ormolecular weight) and/or lowered starch levels. In addition, increasedplant metabolites, such as sugars, could also result from starchalteration or inhibition caused by expression of glycogen biosyntheticenzymes. For example, transgenic potato tubers described herein areobserved to have up to 3-fold increases in free sugar content.

Measurement of specific gravity or free sugar content may be useful todetect modified starch, with other methods, such as HPLC and gelfiltration, also being useful. The glycogen synthase sequence may beemployed as the sole glycogen biosynthetic enzyme or in conjunction withsequences encoding other glycogen biosynthetic enzymes.

In accordance with an additional aspect of the subject invention, thesecond class of reserve polysaccharide modification enzymes includesnovel starch degradation enzymes which permit modification of thecomposition of host plants to increase synthesis of starch degradationproducts. Representative of such enzymes are amylase enzymes such ascyclodextrin glycosyltransferase enzymes, which can provide for theproduction of cyclodextrins from endogenous starch reserves in a varietyof host plants.

As used herein, cyclodextrin glycosyltransferase (CGT) is intended toinclude any equivalent amylase enzyme capable of degrading starch to oneor more forms of cyclodextrin. Considerations for use of a specific CGTin plants for the conversion of starch to cyclodextrin include pHoptimum of the enzyme and the availability of substrate and cofactorsrequired by the enzyme. The CGT of interest should have kineticparameters compatible with the biochemical systems found in the hostplant cell. For example, the selected CGT may compete for starchsubstrate with other enzymes.

The most preferred cyclodextrin forms are the α-, β- or γ- forms,although other higher forms of cyclodextrins, e.g. δ-, ε-, ζ- and η-forms, are also possible. Different CGT enzymes produce α, β, and γ CDsin different ratios. See, Szejtli, J., Cyclodextrin Technology (KluwerAcademic Publications, Boston) (1988), pp. 26-33 and Schmid, G., TIBTECH(1989) 7:244-248. In addition, various CGT enzymes can preferentiallydegrade the starch substrate to favor production of a particularcyclodextrin form. Some CGTs produce primarily β-CDs (Bender, H (1990)Carb. Res. 206:257-267; Kimura et al. (1987) Appl. Microbiol.Biotechnol. 26:149-153), whereas the Klebsiella CGT described in thefollowing examples, produces α- and β-CDs in vitro at a ratio of 20:1when potato starch is used as the substrate (Bender, H. (1990) supra).The use of these different CGTs in transgenic plants could result indifferent CD profiles and thus different utilities. For example,cyclodextrins have been reported as effective in inhibiting apple juicebrowning, with β-cyclodextrins producing better results than either α-or γ-cyclodextrins (Chemistry and Industry, London (1988) 13:410). Inaddition, it has been discovered that in vitro application of β-CDs topotato tuber slices inhibits discoloration, and in vitro application towhole potato tubers prevents a typical blackspot reaction caused bybruising. Additional disclosure concerning cyclydextringlycosyltransferase enzymes can be found in U.S. patent application Ser.No. 07/536,392, filed on Jun. 11, 1990, the complete specification ofwhich is incorporated herein by this reference.

An enzyme relevant to the present invention as including any sequence ofamino acids, such as protein, polypeptide, or peptide fragment, whichdemonstrates the ability to catalyze a reaction involved in themodification of the reserve polysaccharide content of a transformed hostcell.

In one aspect of the invention, the modification will result in thebiosynthesis of glycogen. Thus, a glycogen biosynthetic enzyme of thisinvention will display activity towards a glucan molecule, although itmay have preferential activity towards either ADP- or UDP-glucose. Inplants, ADP-glucose is the preferred donor for starch biosyntheticreactions. Therefore, of particular interest in this invention areglycogen biosynthesis enzymes which also prefer ADP-glucose. Of specialinterest are glycogen biosynthesis enzymes obtainable from bacterialsources. Over 40 species of bacteria synthesize glycogen, includingEscherichia and Salmonella.

Obtaining glycogen biosynthetic enzymes may be accomplished by a varietyof methods known to those skilled in the art. For example, radiolabelednucleic acid probes may be prepared from a known sequence which willbind to, and thus provide for detection of, other sequences. Glycogenbiosynthesis enzymes may be purified and their sequences obtainedthrough biochemical or antibody techniques, polymerase chain reaction(PCR) may be employed based upon known nucleic acid sequences, and thelike.

In another aspect of the invention, the modification will result in theproduction of novel starch degradation products such as, e.g.,cyclodextrins. The structural gene for a selected CGT can be derivedfrom cDNA, from chromosomal DNA or may be synthesized, either completelyor in part. For example, the desired gene can be obtained by generatinga genomic DNA library from a source for CGT, such as a prokaryoticsource, e.g. Bacillus macerans, Bacillus subtilis or, preferably, fromKlebsiella pneumoneae.

Typically, a gene sequence obtainable from the use of nucleic acidprobes will show 60-70% sequence identity between the target sequenceand the given sequence encoding an enzyme of interest. However, lengthysequences with as little as 50-60% sequence identity may also beobtained. The nucleic acid probes may be a lengthy fragment of thenucleic acid sequence, or may also be a shorter, oligonucleotide probe.Oligonucleotide probes can be considerably shorter than the entirenucleic acid sequence encoding a selected enzyme, but should be at leastabout 10, preferably at least about 15, and more preferably at leastabout 20-nucleotides. A higher degree of sequence identity is desiredwhen shorter regions are used as opposed to longer regions. It may thusbe desirable to identify enzyme active sites where amino acid sequenceidentity is high to design oligonucleotide probes for detectinghomologous genes.

When longer nucleic acid fragments are employed as probes (greater thanabout 100 bp), one may screen at lower stringencies in order to obtainsequences from the target sample which have 20-50% deviation (i.e.,50-80 sequence homology) from the sequences used as probe. Hybridizationand washing conditions can be varied to optimize the hybridization ofthe probe to the sequences of interest. Lower temperatures and highersalt (SSC) concentrations allow for hybridization of more distantlyrelated sequences (low stringency). If background hybridization is aproblem under low stringency conditions, the temperature can be raisedeither in the hybridization or washing steps and/or salt content loweredto improve detection of the specific hybridizing sequence. Hybridizationand washing temperatures can be adjusted based on the estimated meltingtemperature of the probe. (See, for example, Beltz, et al. Methods inEnzymology (1983) 100:266-285).

It will be recognized by one of ordinary skill in the art that selectedenzyme sequences of this invention may be modified using standardtechniques of site specific mutation or PCR, or modification of thesequence may be accomplished in producing a synthetic nucleic acidsequence and will still be considered an enzyme nucleic acid sequence ofthis invention. For example, wobble positions in codons may be changedsuch that the nucleic acid sequence encodes the same amino acidsequence, or alternatively, codons can be altered such that conservativeamino acid substitutions result. In either case, the peptide or proteinmaintains the desired enzymatic activity and is thus considered part ofthe instant invention.

A nucleic acid sequence of an enzyme relevant to the present inventionmay be a DNA or RNA sequence, derived from genomic DNA, cDNA, mRNA, ormay be synthesized in whole or in part. The structural gene sequencesmay be cloned, for example, by isolating genomic DNA from an appropriatesource, and amplifying and cloning the sequence of interest using apolymerase chain reaction (PCR). Alternatively, the gene sequences maybe synthesized, either completely or in part, especially where it isdesirable to provide plant-preferred sequences. Thus, all or a portionof the desired structural gene may be synthesized using codons preferredby a selected plant host. Plant-preferred codons may be determined, forexample, from the codons used most frequently in the proteins expressedin a particular plant host species. Other modifications of the genesequences may result in mutants having slightly altered activity. Onceobtained, an enzyme nucleic acid sequence of this invention may becombined with other sequences in a variety of ways.

Often, the sequences associated with reserve polysaccharide modificationare used in conjunction with endogenous plant sequences. By "endogenousplant sequence" is meant any sequence which can be naturally found in aplant cell. These sequences include native (indigenous) plant sequencesas well as sequences from plant viruses or plant pathogenic bacteria,such as Agrobacterium or Rhizobium species that are naturally found andfunctional in plant cells.

In one aspect of this invention, the selected enzyme sequence will bejoined to a sequence encoding a transit peptide or functional portion ofa transit peptide which is capable of providing for intracellulartransport of a heterologous protein to a plastid in a plant host cell.Chloroplasts are the primary plastid in photosynthetic tissues, althoughplant cells are likely to have other kinds of plastids, includingamyloplasts, chromoplasts, and leucoplasts. Transport into amyloplastsis preferred in this invention as these plastids are associated withreserve starch synthesis and storage. Any transit peptide providing forintracellular transport to a plastid is useful in this invention, suchas the transit peptides from the precursor proteins of the small subunitof ribulose bisphosphate carboxylase (RUBISCO), acyl carrier protein(ACP), the waxy locus of maize, or other nuclear-encoded plastidproteins.

In addition to the identified transit peptide portion of a protein, itmay be desirable to include sequences encoding a portion of the matureplastid-targeted protein to facilitate the desired intracellulartransport of the glycogen biosynthetic enzyme. In one embodiment of thisinvention, the transit peptide from the small subunit of RUBISCO isutilized along with 48 bp of sequence encoding the amino terminal 16amino acids of a mature small subunit protein.

Other endogenous plant sequences may be provided in nucleic acidconstructs of this invention, for example to provide for transcriptionof the enzyme sequences. Transcriptional regulatory regions are locatedimmediately 5' to the DNA sequences of the gene of interest, and may beobtained from sequences available in the literature, or identified andcharacterized by isolating genes having a desirable transcriptionpattern in plants, and studying the 5' nucleic acid sequences. Numeroustranscription initiation regions which provide for a variety ofconstitutive or regulatable, e.g. inducible, expression in a plant cellare known. Among sequences known to be useful in providing forconstitutive gene expression are regulatory regions associated withAgrobacterium genes, such as for nopaline synthase (Nos), mannopinesynthase (Mas), or octopine synthase (Ocs), as well as regions codingfor expression of viral genes, such as the 35S and 19S regions ofcauliflower mosaic virus (CaMV). The term constitutive as used hereindoes not necessarily indicate that a gene is expressed at the same levelin all cell types, but that the gene is expressed in a wide range ofcell types, although some variation in abundance is often detectable.Other useful transcriptional initiation regions preferentially providefor transcription in certain tissues or under certain growth conditions,such as those from napin, seed or leaf ACP, the small subunit ofRUBISCO, patatin, zein, and the like.

Sequences to be transcribed are located 3' to the plant transcriptioninitiation region and may be oriented, in the 5'-3' direction, in thesense orientation or the antisense orientation. In the senseorientation, an mRNA strand is produced which encodes the desiredglycogen biosynthetic enzyme, while in antisense constructs, an RNAsequence complementary to an enzyme coding sequence is produced. Thesense orientation is desirable when one wishes to produce the selectedenzyme in plant cells, whereas the antisense strand may be useful toinhibit production of related plant enzymes. Regions of homology havebeen observed, for example, upon comparison of E. coli glgC sequence tothat of a rice ADP glucose pyrophosphorylase. Either method may beuseful in obtaining an alteration in the polysaccharide or dry mattercontent of a plant. The presence of the selected enzyme sequences in thegenome of the plant host cell may be confirmed, e.g., by a Southernanalysis of DNA or a Northern analysis of RNA sequences or by PCRmethods.

In addition to sequences providing for transcriptional initiation in aplant cell, also of interest are sequences which provide fortranscriptional and translational initiation of a desired sequenceencoding a glycogen biosynthetic enzyme. Translational initiationregions may be provided from the source of the transcriptionalinitiation region or from the gene of interest. In this manner,expression of the selected enzyme in a plant cell is provided. Thepresence of the enzyme in the plant host cell may be confirmed by avariety of methods including a immunological analysis of the protein(e.g. Western or ELISA), as a result of phenotypic changes observed inthe cell, such as altered starch content, altered starch branching,etc., or by assay for increased enzyme activity, and the like. Ifdesired the enzyme may be harvested from the plant host cell or used tostudy the effect of the enzyme on plant cell functions, especially inthe plastid organelles.

Other sequences may be included in the nucleic acid construct providingfor expression of the selected enzymes ("expression constructs") of thisinvention, including endogenous plant transcription termination regionswhich will be located 3' to the desired enzyme encoding sequence. In oneembodiment of this invention, transcription termination sequencesderived from a patatin gene are preferred. Transcription terminationregions may also be derived from genes other than those used to regulatethe transcription in the nucleic acid constructs of this invention.Transcription termination regions may be derived from a variety ofdifferent gene sequences, including the Agrobacterium, viral and plantgenes discussed above for their desirable 5' regulatory sequences.

Further constructs are considered which provide for transcription and/orexpression of more than one selected enzyme. For example, one may wishto provide enzymes to plant cells which provide for modification of thestarch synthesized, as well as for an increase or decrease in overallstarch production. Examples of enzymes which may prove useful inmodifying starch structure are those which catalyze reactions involvingUDP- or ADP-glucose, for example glycogen synthase or branching enzyme.However, to provide for increased or decreased starch production, or theproduction of starch degradation products, one may wish to utilizesequences encoding enzymes which catalyze formation of thenucleotide-glucose molecule, such as ADP-glucose pyrophosphorylase inbacteria, or glucose-1-phosphate uridylyltransferase in mammals.Although plants typically utilize ADP-glucose, UDP-glucose may also beuseful.

In providing for transcription and/or expression of the selected enzymesequences, one may wish to limit these enzymes to plant cells whichsynthesize and store reserve starch. Towards this end, one can identifyuseful transcriptional initiation regions that provide for expressionpreferentially in the roots, tubers, seeds, or other starch-containingtissues of a desired plant species. These sequences may be identifiedfrom cDNA libraries using differential screening techniques, forexample, or may be derived from sequences known in the literature. Ofparticular interest in a presently preferred embodiment of the inventionis a transcriptional initiation region from the patatin gene of potato,which demonstrates preferential expression in the potato tuber.Similarly, other promoters which are preferentially expressed in thestarch-containing tissues, such as the zein genes in corn, as opposed toother plant structures are desirable.

In developing the nucleic acid constructs of this invention, the variouscomponents of the construct or fragments thereof will normally beinserted into a convenient cloning vector, e.g. a plasmid, which iscapable of replication in a bacterial host, e.g. E. coli. Numerousvectors exist that have been described in the literature, many of whichare commercially available. After each cloning, the cloning vector withthe desired insert may be isolated and subjected to furthermanipulation, such as restriction, insertion of new fragments ornucleotides, ligation, deletion, mutation, resection, etc. so as totailor the components of the desired sequence. Once the construct hasbeen completed, it may then be transferred to an appropriate vector forfurther manipulation in accordance with the manner of transformation ofthe host cell.

The constructs of this invention providing for transcription and/orexpression of the enzyme sequences of this invention may be utilized asvectors for plant cell transformation. The manner in which nucleic acidsequences are introduced into the plant host cell is not critical tothis invention. Direct DNA transfer techniques, such as electroporation,microinjection or DNA bombardment may be useful. To aid inidentification of transformed plant cells, the constructs of thisinvention may be further manipulated to include plant selectablemarkers. The use of plant selectable markers is preferred in thisinvention as the amount of experimentation required to detect plantcells is greatly reduced when a selectable marker is expressed. Usefulselectable markers include enzymes which provide for resistance to anantibiotic such as gentamicin, hygromycin, kanamycin, and the like.Similarly, enzymes providing for production of a compound identifiableby color change, such as GUS, or luminescence, such as luciferase, areuseful.

An alternative method of plant cell transformation employs plant vectorswhich contain additional sequences which provide for transfer of thedesired enzyme sequences to a plant host cell and stable integration ofthese sequences into the genome of the desired plant host. Selectablemarkers may also be useful in these nucleic acid constructs to providefor differentiation of plant cells containing the desired sequences fromthose which have only the native genetic material. Sequences useful inproviding for transfer of nucleic acid sequences to host plant cells maybe derived from plant pathogenic bacteria, such as Agrobacterium orRhizogenes, plant pathogenic viruses, or plant transposable elements.

When Agrobacterium is utilized for plant transformation, it may bedesirable to have the selected nucleic acid sequences bordered on one orboth ends by T-DNA, in particular the left and right border regions, andmore particularly, at least the right border region. These borderregions may also be useful when other methods of transformation areemployed.

Where Agrobacterium or Rhizogenes sequences are utilized for planttransformation, a vector may be used which may be introduced into anAgrobacterium host for homologous recombination with the T-DNA on theTi- or Ri-plasmid present in the host. The Ti- or Ri-containing theT-DNA for recombination may be armed (capable of causing gallformation), or disarmed (incapable of causing gall formation), thelatter being permissible so long as a functional complement of the virgenes, which encode trans-acting factors necessary for transfer of DNAto plant host cells, is present in the transformed Agrobacterium host.Using an armed Agrobacterium strain can result in a mixture of normalplant cells, some of which contain the desired nucleic acid sequences,and plant cells capable of gall formation due to the presence of tumorformation genes. Cells containing the desired nucleic acid sequences,but lacking tumor genes can be selected from the mixture such thatnormal transgenic plants may be obtained.

In a preferred method where Agrobacterium is used as the vehicle fortransforming host plant cells, the expression or transcription constructbordered by the T-DNA border region(s) will be inserted into a broadhost range vector capable of replication in E. coli and Agrobacterium,there being broad host range vectors described in the literature.Commonly used is pRK2 or derivatives thereof. See, for example, Ditta,et al., (Proc. Nat. Acad. Sci., U.S.A. (1980) 77:7347-7351) and EPA 0120 515, which are incorporated herein by reference. Alternatively, onemay insert the sequences to be expressed in plant cells into a vectorcontaining separate replication sequences, one of which stabilizes thevector in E. coli, and the other in Agrobacterium. See, for example,McBride and Summerfelt (Plant Mol. Biol. (1990) 14:269-276), wherein thepRiHRI (Jouanin, et al., Mol. Gen. Genet. (1985) 201:370-374) origin ofreplication is utilized and provides for added stability of theexpression vectors in host Agrobacterium cells.

Utilizing vectors such as those described above, which can replicate inAgrobacterium is preferred. In this manner, recombination of plasmids isnot required and the host Agrobacterium vir regions can supplytransacting factors required for transfer of the T-DNA borderedsequences to plant host cells.

In general, the plant vectors of this invention will contain theselected enzyme sequence(s), alone or in combination with transitpeptides, and endogenous plant sequences providing for transcription orexpression of these sequences in a plant host cell. The plant vectorscontaining the desired sequences may be employed with a variety of plantcells, particularly plants which produce and store reserve starch.Plants of interest include, but are not limited to plants which have anabundance of starch in the seed, such as corn (e.g. Zea mays), cerealgrains (e.g. wheat (Triticum spp.), rye (Secale cereale), triticale(Triticum aestium×Secale cereale hybrid), etc.), waxy maize, sorghum(e.g. Sorghum bicolor) and rice (e.g. Oryza sativa), in the rootstructures, such as potato (e.g., Irish (Solanum tuberosum), Sweet(Ipomoea batatas), and yam (Discorea spp.)), tapioca (e.g. cassava(Manihot esculenta)) and arrowroot (e.g., Marantaceae spp., Cycadaceaespp., Cannaceae spp., Zingiberaceae spp., etc.), or in the stem, such assago (e.g. Palmae spp., Cycadales spp.). Starch is also found inbotanical fruits, including for example tomato, apple and pear.

Also considered part of this invention are plants containing the nucleicacid sequences of this invention, and following from that, plantscontaining the selected enzymes as the result of expression of thesequences of this invention in plant cells or having a decreasedexpression of a native enzyme. Methods of regenerating whole plants fromplant cells are known in the art, and the method of obtainingtransformed and regenerated plants is not critical to this invention. Ingeneral, transformed plant cells are cultured in an appropriate medium,which may contain selective agents such as antibiotics, where selectablemarkers are used to facilitate identification of transformed plantcells. Once callus forms, shoot formation can be encouraged by employingthe appropriate plant hormones in accordance with known methods and theshoots transferred to rooting medium for regeneration of plants. Theplants may then be used to establish repetitive generations either fromseed or using vegetative propagation techniques.

Of particular interest are plant parts, e.g. tissues or organs, (andcorresponding cells) which form and store reserve starch, such as roots,tubers, and seeds. Of more particular interest are potato tuberscontaining the selected enzymes. It can be recognized that themodification of enzymes in plants may also result in desirablealterations in the plant cells or parts. These alterations may includemodification of dry matter content, free sugar content or of starchcontent and/or structure, or modification of specific gravity. The novelplant cells or plant parts can thus be harvested and used for isolationof the altered material.

Once the cells are transformed, transgenic cells may be selected bymeans of a marker associated with the expression construct. Theexpression construct will usually be joined with such a marker to allowfor selection of transformed plant cells, as against those cells whichare not transformed. As before, the marker will usually provideresistance to an antibiotic, e.g., kanamycin, gentamicin, hygromycin,and the like, or an herbicide, e.g. glyphosate, which is toxic to plantcells at a moderate concentration.

After transformation, the plant cells may be grown in an appropriatemedium. In the case of protoplast transformations, the cell wall will beallowed to reform under appropriate osmotic conditions. In the case ofseeds or embryos, an appropriate germination or callus initiation mediumwould be employed. For transformation in explants, an appropriateregeneration medium is used.

The callus which results from transformed cells may be introduced into anutrient medium which provides for the formation of shoots and roots,and the resulting plantlets planted and allowed to grow to seed. Duringthe growth, tissue may be harvested and screened for the presence ofexpression products of the expression construct. After growth, thetransformed hosts may be collected and replanted. One or moregenerations may then be grown to establish that the enzyme structuralgene is inherited in Mendelian fashion.

The ability to modify the composition of a host plant offers potentialmeans to alter properties of the plant produce, such as, e.g., by thereplacement of endogenous starch with oligosaccharides comprisingglucopyranose units. These oligosaccharides, cyclodextrins for example,may then be purified away from the other plant components. For example,by modifying crop plant cells by introducing a functional structuralgene expressing a selected enzyme, one can provide a wide variety ofcrops which have the ability to produce starch degradation products, anddesirably such production will be effected without damaging theagronomic characteristics of the host plant. In this manner, substantialeconomies can be achieved in labor and materials for the production ofstarch degradation products, while minimizing the detrimental effects ofstarch degradation on the host plants.

Preferably, the activity of the starch degradation enzyme will belocalized in the starch storage organelles, tissues or regions of thehost plant, e.g., the amyloplast of a host potato tuber. The structuralgene will manifest its activity by mediating the production ofdegradation products in at least one portion of the genetically modifiedhost plant.

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

EXPERIMENTAL

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); mM (millimolar); μM(micromolar); N (Normal); mol (moles); mmol (millimoles); μmol(micromoles); nmol (nanomoles); kg (kilograms); g (grams); mg(milligrams); μg (micrograms); ng (nanograms); L (liters); ml(milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm(micrometers); nm (nanometers); V (volts); μF (microfarads) and °C.(degrees Centigrade).

In order to demonstrate the practice of the present invention inutilizing reserve polysaccharide biosynthetic enzymes, the followingexamples convey an embodiment for the biosynthesis of glycogen.

EXAMPLE 1 Cloning of Glycogen Biosynthetic Enzyme Genes

A. Cloning and Sequencing of a GlgA Gene From E. coli

Total genomic DNA is prepared from E. coli K12 618 (Leung et al., J. ofBacteriology (1986) 167:82-88) by growing a 5 ml culture in ECLB(Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, (1982)Cold Spring Harbor, N.Y.) overnight at 37° C. The bacteria are pelletedby centrifugation for 10 minutes at 4500×g, the supernatant isdiscarded, and the pellet is resuspended in 2.5 ml of 10 mM Tris, 1 mMEDTA buffer. To this suspension is added 500 μl of a 5 mg/ml Pronase®protease (Calbiochem Brand Biochemicals; La Jolla, Calif.) solution and2 ml of 2% lauryl sulfate, sodium salt (Sigma; St. Louis, Mo.), withgentle mixing, and the suspension is incubated at 37° C. for 50 minutes.A clear solution indicates that the bacteria have lysed. The solution isthen extracted with 5 ml phenol, then 5 ml phenol:chloroform:isoamylalcohol (25:24:1), followed by 5 ml chloroform. Nucleic acids areprecipitated from the aqueous phase with 1/10 volume of 3M sodiumacetate and two volumes of 100% ethanol, and the tube is incubated atroom temperature for 1 hour. Nucleic acids are removed from solution andresuspended in 1 ml water. A second ethanol precipitation is performedand the nucleic acids are resuspended in 200 μl of 10 mM Tris, 1 mM EDTAbuffer.

Synthetic oligonucleotides, str1 and str2, corresponding to sequencesflanking the 1.4 kb glgA (glycogen synthase--EC 2.4.1.21) gene of E.coli (Kumar et al., J. of Biol. Chemistry (1986) 261:16256-16259) andcontaining restriction sites for BglII (str1) and SalI (str2) aresynthesized on an Applied Biosystems 380A DNA synthesizer (Foster City,Calif.) in accordance with the manufacturer's instructions.

The nucleic acid preparation of E. coli is treated with RNAse and theDNA is used as a template in a polymerase chain reaction (PCR) with str1and str2 as primers. A Perkin-Elmer/Cetus (Norwalk, Conn.) thermalcycler is used with the manufacturer's reagents and in accordance withthe manufacturer's instructions. The reaction mixture contains 41.5 μlH₂ O, 10 μl 10× reaction buffer, 16 μl dNTP's 1.25 mM dCTP, dATP, dGTP &dTTP!, 5 μl str1 (20 mM), 5μl str2 (20 mM), 22 μl total E. coli DNA(0.05 μg/μl), and 0.5 μl Taq polymerase. The reaction is performed for15 cycles with melting (denaturation) for 1 minute at 94° C., annealing(hybridization) for 2 minutes at 37° C. and chain elongation for 3minutes at 72° C. The reaction is then performed for an additional 10cycles with melting for 1 minute at 94° C., annealing for 2 minutes at37° C. and chain elongation at 72° C. for 3 minutes 15 seconds initiallyand increasing the time by 15 seconds each cycle so that the last cycleis 5 minutes 45 seconds.

The resulting PCR products (˜1.4 kb) are digested with BglII and SalIand ligated into a SalI and BglII digest of pCGN789, a pUC based vectorsimilar to pUC119 with the normal polylinker replaced by a syntheticlinker which contains the restriction digest sites EcoRI, SalI, BglII,PstI, XhoI, BamHI, and HindIII. The ligated DNA is transformed into E.coli DH5α. The transformed cells are plated on ECLB containingpenicillin (300 mg/L), IPTG and X-Gal (Vieira and Messing, Gene (1982)19:259-268). White colonies are picked to ECLB containing penicillin(300 mg/L) and flooded with I₂ /KI (0.2% I₂ in 0.4% KI). Clonesproducing a brown color, which indicates excess starch production, areselected. One clone, glgA-2, is selected and the DNA and translatedamino acid sequences are determined (see, FIGS. 1 and 2 and SEQ ID NOS:1-2). The DNA sequence is 98% homologous to the published sequence(Kumar et al, supra) and 96% homologous at the amino acid level.

B. Cloning and Sequencing of a GlgC Gene From E. coli

Synthetic oligonucleotides, glgC1 and glgC2, corresponding to sequencesflanking the 1.3 kb glgC (ADP-glucose pyrophosphorylase - EC 2.7.7.27)gene of E. coil (Baecker et al., J. of Biol. Chemistry (1983)258:5084-5088) and containing restriction sites for BglII (glgC1) andSalI (glgC2) are synthesized on an Applied Biosystems 380A DNAsynthesizer (Foster City, Calif.) in accordance with the manufacturer'sinstructions.

Total genomic DNA is prepared from E. coli K12 618 as described above.The nucleic acid preparation of E. coli is treated with RNAse and theDNA is used as a template in a polymerase chain reaction (PCR) withglgC1 and glgC2 as primers. A Perkin-Elmer/Cetus (Norwalk, Conn.)thermal cycler is used with the manufacturer's reagents as describedabove.

The resulting PCR products (˜1.3 kb) are digested with BglII and SalIand ligated into a SalI and BglII digest of pCGN789 (described above).The ligated DNA is transformed into E. coli DH5α, and the transformedcells are plated as described above. Clones producing excess starch areselected as described above. One clone, pGlgC-37, is selected and theDNA sequence (SEQ ID NO: 3) determined (see, FIG. 3). The DNA sequenceis 99% homologous to the published sequence (Baecker et al, supra) ofglgC from E. coli K-12. The glgC from E. coli 618 is a mutant and theamino acid sequence of this mutant differs from that of E. coli K-12 atfive amino acids (Lee et al., Nucl. Acids Res. ; (1987) 15:10603). Thetranslated amino acid sequence of pGlgC-37 differs from that of the glgCfrom E. coli 618 at a single amino acid; the asparagine (Asn) atposition 361 of the E. coli 618 mutant is an aspartate (Asp) in thetranslated amino acid sequence of pGlgC-37 (FIG. 3).

EXAMPLE 2 Attachment of Glycogen Genes to SSU Leader Sequence

A. Construction of SSU+aroA Transit Peptide

Plasmid pCGN1132 contains a 35S promoter, ribulosebisphosphatecarboxylase small subunit (5'-35S-SSU) leader from soybean plus 48 bp ofmature small subunit (SSU) gene from pea, and aroA sequence (the genelocus which encodes 5-enolpyruvyl-3-phosphoshikimate synthetase (EC2.5.1.19)). It is prepared from pCGN1096, a plasmid containing a hybridSSU gene, which carries DNA encoding mature SSU protein from pea, andSstI and EcoRI sites 3' of the coding region (used in the preparation ofpCGN1115, a plasmid having a 5'-35S-SSU+48-aroA-tml-3' sequence) andpCGN1129, (a plasmid having a 35S promoter in a chloramphenicolresistance gene (Cam^(r)) backbone).

Construction of pCGN1096

The aroA moiety of pCGN1077 is removed by digestion with SphI and SalI.(The construction of pCGN1077 and other constructs hereunder aredescribed in detail in copending U.S. application Ser. No. 06/097,498,filed Sep. 16, 1987, which is hereby incorporated by reference). In itsplace is cloned the region coding for the mature pea SSU protein, as anSphI-PstI fragment, which is then excised with SphI and SalI. Theresulting plasmid, pCGN1094, codes for a hybrid SSU protein having thetransit peptide of the soybean clone, and the mature portion of the peaclone and contains SstI and EcoRI sites 3' of the coding region. TheHindIII to BamHI region of transposon Tn6 (Jorgensen et al., Mol. Gen.Genet. (1979) 177:65) encoding the kanamycin resistance gene (Kan^(r))is cloned into the same sites of pBR322 (Bolivar et al., Gene (1977)2:95-133) generating pDS7. The BglII site 3' of the Kan^(r) gene isdigested and filled in with the large fragment of E. coli DNA polymerase1 and deoxynucleotides triphosphate. An SstI linker is ligated into theblunted site, generating plasmid pCGN1093. Plasmid pPMG34.3 is digestedwith SalI, the site filled in as above and EcoRI linkers are ligatedinto the site resulting in plasmid pCGN1092. The latter plasmid isdigested with SstI and SmaI and the Kan^(r) gene excised from pCGN1093with SstI and SmaI is ligated in, generating pCGN1095. The Kan^(r) andaroA genes are excised as a piece from pCGN1095 by digestion with SstIand EcoRI and inserted into the SstI and EcoRI sites of pCGN1094,producing pCGN1096. Summarizing, pCGN1096 contains (5'-3') the followingpertinent features: The SSU gene--a polylinker coding for PstI, SalI,SstI, and KpnI--the Kan^(r) gene--SmaI and BamHI restriction sites--thearoA gene without the original ATG start codon.

Construction of pCGN1115

Plasmid pCGN1096 is digested to completion with SalI and then digestedwith exonuclease Bal31 (BRL; Gaithersburg, Md.) for 10 minutes, thusdeleting a portion of the mature SSU gene. The resulting plasmid is thendigested with SmaI to eliminate the Kan^(r) gene and provide blunt ends,recircularized with T4 DNA ligase and transformed into E. coli LC3(Comai et al., Science (1983) 221:370-371), an aroA mutant. DNA isolatedfrom aroA⁺ and Kan^(r) colonies is digested with BamHI and SphI andligated with BamHI- and SphI-digested M13mp18 (Norrander et al., Gene(1983) 26:101-106 and Yanisch-Perron et al., Gene (1985) 33:103-119) DNAfor sequencing. Clone 7 has 48bp of the mature SSU gene remaining andthe 3'-end consists of phe-glu-thr-leu-ser (SEQ ID NO: 1). Clone 7 istransformed into E. coli strain 71-18 (Yanish-Perron et al. (1985)supra) and DNA isolated from transformants is digested with SphI andClaI to remove the 0.65 kb fragment containing the 48 bp of matureprotein and the 5'-end of the aroA gene. Plasmid pCGN1106 (Comai, L. etal., J. Biol. Chem. (1988) 263:15104-15109) is also digested with SphIand ClaI and the 6.8 kb isolated vector fragment is ligated with the0.65 kb fragment of clone 7 to yield pCGN1115(5'-35S-SSU+48-aroA-tml-3').

Construction of pCGN1129

The 7.2 kb plasmid pCGN1180 (35S-SSU+70-aroA-ocs3') (Comai et al. (1988)supra) and the 25.6 kp plasmid pCGN594 (LB-Gent^(r) -ocs5'-Kan^(r) -ocs3'-RB) (construction of pCGN594 is described in co-pending U.S.application Ser. No. 07/382,802, filed Jul. 19, 1989) are digested withHindIII and ligated together to yield the 32.8 kb plasmid pCGN1109(LB-Gent^(r) -35S-SSU+70-aroA-ocs3'-ocs5'-Kan^(r) -ocs3 '-RB).

Plasmid pCGN1109 is digested with EcoRI to delete an internal 9.1 kbfragment containing the SSU leader plus 70 bp of the mature SSU gene,the aroA gene and its ocs3' terminator, the Amp^(r) backbone frompCGN1180 and ocs5'-Kan^(r) -ocs3' from pCGN594. The EcoRI digest ofpCGN1109 is then treated with Klenow fragment to blunt the ends, and anXhoI linker (dCCTCGAGG) (New England Biolabs.; Beverly, Mass.) isligated in, yielding pCGN1125 (LB-35S-RB).

Plasmid pCGN1125 is digested with HindIII and BglII to delete the 0.72kb fragment of the 35S promoter. This digest is ligated with HindIII-and BamHI-digested Cam^(r) vector, pCGN786 (described in co-pending U.S.application Ser. No. 07/382,803, filed Jul. 19, 1989). The resulting3.22 kb plasmid, pCGN1128, contains the 35S promoter with a 3'multilinker in a Cam^(r) backbone.

Plasmid pCGN1128 is digested with HindIII, treated with Klenow fragmentto blunt the ends and ligated with BglII linkers to yield pCGN1129, thuschanging the HindIII site located 5' to the 35S promoter into a BglIIsite.

B. Transit Peptide Joined to GlgA Gene Plasmid pCGN1115 is digested withSalI to remove a 1.6kb fragment containing the SSU leader plus 46bp ofthe mature SSU gene and the aroA gene. An XhoI digest of pCGN1129 opensthe plasmid 3' to the 35S promoter. Ligation of these two digests yieldsthe 4.8 kb plasmid pCGN1132, containing 5'-35S-SSU leader plus 48 bp ofmature SSU-aroA.

Plasmid pGlgA-2 is digested with BglII and SalI and ligated to pCGN1132that has been digested with BamHI and SalI. A clone containing5'-35S-SSU+48bp-glgA-3' is selected and designated pCGN1439.

C. Transit Peptide Joined to GlgC Gene

Plasmid pGlgC-37 is digested with BglII and SalI and ligated to pCGN1132that has been digested with BamHI and SalI. A clone containing5'-35S-SSU+48bp-glgC-3' is selected and designated pCGN1440.

EXAMPLE 3 Cloning of Patatin Regulatory Regions and Preparation ofPatatin-5'-nos-3' Expression Cassettes

This example describes the cloning of a patatin-5' regulatory regionfrom potato and the preparation of patatin-5'-nos-3' expression cassettepCGN2143.

Genomic DNA is isolated from leaves of Solanum tuberosum var. Kennebecas described in Dellaporta et al., Plant Mol. Biol. Reporter (1983)1(4):19-21), with the following modifications: approximately 9 g freshweight of leaf tissue is ground, a polytron grinding is not performedand in the final step the DNA is dissolved in 300 μl of 10 mM Tris, 1 mMEDTA, pH 8.

A synthetic oligonucleotide, pat1, containing digestion sites for NheI,PstI and XhoI with 24 bp of homology to the 5'-region of a 701 bpfragment (coordinates 1611 to 2313) 5' to a class I patatin gene,isolated from Solanum tuberosum var. Maris Piper (Bevan et al., NAR(1986) 14:4625-4638), is synthesized (Applied BioSystems 380A DNAsynthesizer). A second synthetic oligonucleotide, pat2, containingdigestion sites for BamHI and SpeI with 25 bp of homology to the 3'region of the 703 bp piece is also synthesized.

Using the genomic potato DNA as a template, and pat1 and pat2 asprimers, a polymerase chain reaction (PCR) is performed in aPerkin-Elmer/Cetus thermal cycler with the manufacturer's reagents andin accordance with the manufacturer's instructions. The reactioncontains 62.5 μl H₂ O, 10 μl 10× Reaction buffer, 16 μl dNTP's 1.25 mMdCTP, dATP, dGTP & dTTP!, 5 μl pat1 (20 mM), 5 μl pat2 (20mM), 1 μlpotato genomic DNA (3 μg/μl), 0.5 μl Tag polymerase. The PCR isperformed for 25 cycles with melting for 1 minute at 94° C., annealingfor 2 minutes at 37° C. and chain elongation for 3 minutes at 72° C. Theresulting PCR product fragments (approximately 700 bp) are digested withNheI and BamHI. Plasmid pCGN1586N (5-D35S-TMVΩ'-nos-3'; pCGN1586(described below) having a NheI site 5' to the 35S region) is digestedwith NheI and BamHI to delete the D35S-Ω' fragment. Ligation ofNheI-BamHI digested pCGN1586N, which contains the nos-3' region, and thePCR fragments yields a patatin-5'-nos3' cassette with SpeI, BamHI, SalIand SstI restriction sites between the 5' and 3' regions for insertionof a DNA sequence of interest.

The 5' region of a clone, designated pCGN2143 is sequenced. PlasmidpCGN2143 has a Kennebec patatin-5' region that is 702 bp in length and99.7% homologous to the native sequence (as reported by Bevan (1986)supra).

Synthetic oligonucleotides, pat5 and pat6, are prepared as describedabove. Pat5 and pat6 contain complementary sequences which contain therestriction digest sites NheI, XhoI and PstI. Pat5 and pat6 are annealedto create a synthetic linker. The annealed linker is ligated to pCGN2143that has been linearized with EcoRI and treated with Klenow polymeraseto generate blunt ends. A plasmid, pCGN2162 which has the followingrestriction sites at the 3' end of nos is selected:5'-EcoRI-NheI-XhoI-PstI-EcoRI.

Construction of pCGN1586/1586N

Plasmid pCGN2113 (6.1 kb) contains a double-35S promoter (D35S) and thetml-3' region with multiple cloning sites between them, contained in apUC-derived plasmid backbone bearing an ampicillin resistance gene(Ampr). The promoter/tml cassette is bordered by multiple restrictionsites for easy removal. Plasmid pCGN2113 is digested with EcoRI andSacI, deleting the 2.2 kb tml-3' region. Plasmid pBI221.1 (Jefferson, R.A., Plant Mol. Biol. Reporter (1987) 5:387-405) is digested with EcoRIand SacI to delete the 0.3 kb nos-3' region. The digested pCGN2113 andpBI221.1 DNAs are ligated together, and the resultant 4.2 kb recombinantplasmid with the tml-3' of pCGN2113 replaced by nos-3' is designatedpCGN1575 (5'-D35S-nos-3').

Plasmid pCGN1575 is digested with SphI and XbaI, blunt ends generated bytreatment with Klenow fragment, and the ends are ligated together. Inthe resulting plasmid, pCGN1577, the SphI, PstI, SalI and XbaI sites 5'of the D35S promoter are eliminated.

Plasmid pCGN1577 is digested with EcoRI, the sticky ends blunted bytreatment with Klenow fragment, and synthetic BglII linkers(d(pCAGATCTG) New England Biolabs, Inc.; Beverly, Mass.) are ligated in.A total of three BglII linkers are ligated into the EcoRI site creatingtwo PstI sites. The resulting plasmid, termed pCGN1579 (D35S-nos-3'),has a 3' polylinker consisting of 5'-EcoRI, BglII, PstI, BglII, PstI,BglII, EcoRI-3¹.

A tobacco mosaic virus omega' (TMVΩ) region (Gallie et al., NAR (1987)15(21) :8693-8711) with BglII, NcoI, BamHI, SalI and SacI restrictionsites: ##STR1## is synthesized on an Applied Biosystems® 380A DNAsynthesizer and digested with BglII and SacI. Plasmid pCGN1577 isdigested with BamHI and SacI and the synthetic TMVΩ is ligated inbetween the 5'-D35S and nos-3' regions. The resulting plasmid isdesignated pCGN1586 (5'-D35S-TMVΩ'-nos-3'). Plasmid pCGN1586N is made bydigesting pCGN1586 with HindIII and filling in the 5' overhang withKlenow fragment, thus forming a NheI site 5' to the D35S region.

Plasmid pCGN2143 is also described in co-pending U.S. application Ser.No. 07/536,392 filed Jun. 11, 1990, which is hereby incorporated byreference.

EXAMPLE 4 Preparation of Binary Vectors

This example describes the construction of a binary vector containing:(1) the patatin-5' region from Solanum tuberosum var. Kennebec, (2) DNAencoding a transit peptide from soybean RuBisCo SSU protein, (3) 48 bpof DNA encoding 16 amino acids of mature RuBisCo SSU protein from pea,(4) the glgA coding region from E. coli 618 and (5) the nos-3' region.

A. GlgA Construct

Plasmid pCGN2162 prepared as described in Example 3 is digested withSpeI and SalI, opening the plasmid between the patatin-5' region andnos-3' region. Plasmid pCGN1439 (described in Example 2) is digestedwith XbaI and SalI and ligated with pCGN2162 to yield pCGN1454. PlasmidpCGN1454 consists of 5' -Kennebec patatin-SSU+48-glgA-nos3'.

Plasmid pCGN1454 is digested with XhoI and treated with Klenowpolymerase to generate blunt ends. Plasmid pCGN1557 is digested withXbaI and treated with Klenow polymerase to generate blunt ends. Thefragments resulting from the digests are ligated together. Thetransformation is plated onto ECLB containing gentamycin, IPTG andX-Gal. White colonies are picked and screened for ampicillinsensitivity. Gent^(r), Amp^(s) clones are analyzed and two clones areselected. Plasmid pCGN1457 has the 5'patatin-SSU+48bp-glgA-nos3'inserted into pCGN1557 such that it transcribes in the oppositedirection from the 35S-Kan^(r) -tml gene. Plasmid pCGN1457B has the5'patatin-SSU+48bp-glgA-nos3' inserted into pCGN1557 such that ittranscribes in the same direction as the 35S-Kan^(r) -tml gene.

B. GlgC Construct

Plasmid pCGN2162 prepared as described in Example 3 is digested withSpeI and SalI, opening the plasmid - between the patatin-5' region andnos-3' region. Plasmid pCGN1440 (described in Example 2) is digestedwith XbaI and SalI and ligated with pCGN2162 to yield pCGN1453. PlasmidpCGN1453 consists of 5'-Kennebec patatin-SSU+48-glgC-nos3'.

Plasmid pCGN1453 is digested with PstI and ligated to a PstI digest ofpCGN1557. The transformation is plated as described above and coloniesare screened for ampicillin sensitivity. Gent^(r), Amp^(s) clones areanalyzed and one clone, pCGN1455, is selected. Plasmid pCGN1455 has the5'patatin-SSU+48bp-glgC-nos3' inserted into pCGN1557 such that ittranscribes in the same direction as the 35S-Kan^(r) -tml gene.

C. Construction of pCGN1557

Plasmid pCGN1557 (McBride and Summerfelt, Plant Mol. Biol. (1990)14(27):269-276) is a binary plant transformation vector containing theleft and right T-DNA borders of Agrobacterium tumefaciens octopineTi-plasmid pTiA6 (Currier and Nester, J. Bact. (1976) 126:157-165), thegentamicin resistance gene (Gen^(r)) of pPH1JI (Hirsch and Beringer,Plasmid (1984) 12:139-141), an Agrobacterium rhizogenes Ri plasmidorigin of replication from pLJbB11 (Jouanin et al., Mol. Gen. Genet.(1985) 201:370-374), a 35S promoter-Kan^(r) -tml-3' region capable ofconferring kanamycin resistance to transformed plants, a ColE1 origin ofreplication from pBR322 (Bolivar et al. (1977) supra) and a lacZ'screenable marker gene from pUC18 (Yanisch-Perron et al., (1985) supra).The construction of pCGN1557 is also described in co-pending U.S.application Ser. No. 07/494,722, filed Mar. 16, 1990.

EXAMPLE 5 Preparation of Transgenic Plants

This example describes the transformation of Agrobacterium tumefacienswith glycogen biosynthetic enzyme gene nucleic acid constructs inaccordance with the present invention and the cocultivation of these A.tumefaciens with plant cells to produce transgenic plants containing theglycogen constructs.

A. Transformation of Agrobacterium tumefaciens

Cells of Agrobacterium tumefaciens strain 2760 (also known as LBA4404,Hoekema et al., Nature (1983) 303:179-180) are transformed with binaryvectors, such as pCGN1457, pCGN1457B and pCGN1455 (as described inExample 4) using the method of Holsters, et al., (Mol. Gen. Genet.,(1978) 163:181-187). The transformed A. tumefaciens are then used in theco-cultivation of plants.

The Agrobacterium are grown on AB medium (K₂ HPO₄ 6 g/L, NaH₂ PO₄.H₂ O2.3 g/L, NH₄ Cl 2 g/L, KCl 3 g/L, glucose 5 g/L, FeSO₄ 2.5 mg/L, MgSO₄246 mg/L, CaCl₂ 14.7 mg/L, 15 g/L agar), plus 100 μg/L gentamycinsulfate and 100 μg/L streptomycin sulfate for 4-5 days. Single coloniesare inoculated into 10 ml of MG/L broth (per liter: 5 g mannitol, 1 gL-Glutamic acid or 1.15 g sodium glutamate, 0.5 g KH₂ PO₄, 0.10 g NaCl,0.10 g MgSO₄ 0.7H₂ O, 1 μg biotin, 5 g tryptone, 2.5 g yeast extract;adjust pH to 7.0) and are incubated overnight in a shaker at 30° C. and180 rpm. Prior to co-cultivation, the Agrobacterium culture iscentrifuged at 12,000×g for 10 minutes and resuspended in 20 ml of MSmedium (#510-1118, Gibco; Grand Island, N.Y.).

B. Cocultivation with Potato Cells

Feeder plates are prepared by pipetting 0.5 ml of a tobacco suspensionculture (-10⁶ cells/ml) onto 0.8% agar co-cultivation medium, containingMurashige and Skoog salts (#510-117, Gibco; Grand Island, N.Y.),thiamine-HCl (1.0 mg/L), nicotinic acid (0.5 mg/L), pyridoxine HCl (0.5mg/L), sucrose (30 g/L), zeatin riboside (5 μM),3-indoleacetyl-DL-aspartic acid (3 μM), pH 5.9. The feeder plates areprepared one day in advance and incubated at 25° C. A sterile 3 mmfilter paper disk is placed on top of the tobacco cells after thesuspension cells have grown for one day.

Tubers of Solanum tuberosum var Russet Burbank between the age of 1 and6 months post harvest are peeled and washed in distilled water. Allsubsequent steps are carried out in a flow hood using steriletechniques. For surface sterilization, tubers are immersed in a solutionof 10% commercial bleach (sodium hypochlorite) with 2 drops of Ivory®liquid soap per 100 ml for 10 minutes. Tubers are rinsed six times insterile distilled water and kept immersed in sterile liquid MS medium(#1118, Gibco; Grand Island; N.Y.) to prevent browning. Tuber discs (1-2mm thick) are prepared by cutting columns of potato tuber with a ˜1 cmin diameter cork borer and slicing the columns into discs of the desiredthickness. Discs are placed into the liquid MS medium culture of thetransformed Agrobacterium tumefaciens containing the binary vector ofinterest (1×10⁷ -1×10⁸ bacteria/ml) until thoroughly wetted. Excessbacteria are removed by blotting discs on sterile paper towels. Thediscs are co-cultivated with the bacteria for 48 hours on the feederplates and then transferred to regeneration medium (co-cultivationmedium plus 500 mg/L carbenicillin and 100 mg/L kanamycin). In 3 to 4weeks, shoots develop from the discs.

When shoots are approximately 1 cm, they are excised and transferred toa 0.8% agar rooting medium containing MS salts, thiamine-HCl (1.0 mg/L),nicotinic acid (0.5 mg/L), pyridoxine-HCl (0.5 mg/L), sucrose (30 g/L),carbenicillin (200 mg/L) and kanamycin (100-200 mg/L) pH 5.9. Plants arerooted two times with at least one rooting taking place on rootingmedium with the higher level of kanamycin (200 mg/L). Plants which haverooted twice are then confirmed as transformed by performing NPTII blotactivity assays (Radke, S. E. et al, Theor, Appl. Genet. (1988)75:685-694). Plants which are not positive for NPTII activity arediscarded.

EXAMPLE 6 Analysis of Tubers from Transformed Potato Plants

In this Example, measurement of specific gravity in tubers fromtransgenic potato plants is described.

Rooted plants, transformed as described in Example 5, are cut into fivesections at the internodes and each section is rooted again, also asdescribed in Example 5. The newly rooted plants are transplanted fromrooting medium to soil and placed in a growth chamber (21° C., 16 hourdays with 250-300 μE/m² /sec). Soil is prepared as follows: For about340 gallons, combine 800 pounds 20/30 sand (approximately 14 cubicfeet), 16 cubic feet Fisons Canadian Peat Moss, 16 cubic feet #3vermiculite, and approximately 4.5 pounds hydrated lime in a Gleasonmixer. The soil is steamed in the mixer for two hours; the mixer mixesfor about 15 seconds at intervals of fifteen minutes over a period ofone hour to ensure even heating throughout the soil. During and afterthe process of steaming, the soil reaches temperatures of at least 180°F. for one hour. The soil is left in the mixer until the next day. Atthat time, hydrated lime is added, if necessary, to adjust the pH torange between 6.30 and 6.80.

The relative humidity of the growth chamber is maintained at 70-90% for2-4 days, after which the humidity is maintained at 40-60%. When plantsare well established in the soil, after approximately two weeks, theyare transferred to a greenhouse. In the greenhouse, plants are grown in6.5 inch pots in a soil mix of peat:perlite:vermiculite (11:1:9), at anaverage temperature of 24° C. day/12° C. night. Day length isapproximately 12 hours and light intensity levels vary fromapproximately 600 to 1000 μE/m² / sec.

Tubers from each plant are harvested and washed 14 weeks after transferto the greenhouse. Immediately after harvest, three to five uniformlysized tubers from each pot are weighed and their specific gravitydetermined. In determining specific gravity, the tubers from each plantare first collectively weighed in air and then collectively weighed inwater. Specific gravity is determined, where x=the weight of tubers inair and y=the weight of tubers in water, as x/(x-y).

In general, the specific gravities of tubers from five replicates ofplants transformed with the glgA constructs (pCGN1457 and pCGN1457B) andof tubers from control plants are determined. Control plants includeregenerated non-transformed potato plants and transgenic potato plantswhich lack the glgA constructs. Controls are subjected to thetransformation and regeneration culture and growth conditions describedabove in production of glgA transformed plants. To compare values fromeach tuber sample, the specific gravity measurements are converted toreflect % total solids content of tubers. Percent total solids iscalculated as (specific gravity)×(199.63)-194.84 (Porter, et al., Am.Pot. J. (1964) 41:329-336). Differences are detected in percent totalsolids as determined for tubers from several of the glgA transformedplants as compared to tubers from control plants.

Results are presented in Table 1 which represent average specificgravity of tubers of 5 replicate plants, except as otherwise indicated.Specific gravity measurements are determined for three to five uniformlysized tubers from each plant and the measurements of the tubers from thereplicate plants are then averaged to determine average specific gravity(SpGr) of tubers for each transformation event. Values for one set oftransformed control plants (Tx) and one set of untransformed/regeneratedcontrol plants (Rg) for each construct are shown at the top of theirrespective columns. Transformed control plants are transformed with anon-carbohydrate-related gene.

                  TABLE 1                                                         ______________________________________                                        Average Specific Gravity Measurements                                         Event        SpGr      Event        SpGr                                      ______________________________________                                        Controls               Controls                                               Tx           1.079     Tx           1.083                                     Rg           1.081     *Rg          1.077                                     Transformed Plants Transformed Plants                                         1457-3       1.073     1457B-3      1.062                                     1457-4       1.060     1457B-4      1.075                                     1457-6       1.076     1457B-5      1.073                                     1457-7       1.080     1457B-7      1.066                                     1457-8       1.077     1457B-8      1.066                                     1457-9       1.067     1457B-9      1.063                                     1457-10      1.083     1457B-10     1.075                                     1457-11      1.065     1457B-12     1.065                                     1457-12      1.066     1457B-13     1.058                                     1457-13      1.080     *1457B-15    1.053                                     1457-14      1.062     1457B-16     1.075                                     1457-15      1.064     1457B-17     1.053                                     1457-16      1.068     1457B-18     1.068                                     1457-17      1.069     1457B-21     1.081                                     1457-18      1.060     1457B-22     1.067                                     1457-19      1.069     1457B-23     1.069                                     1457-20      1.066     1457B-24     1.068                                     1457-22      1.068                                                            ______________________________________                                         *Only 4 replicate plants are available for these samples.                

It is readily apparent from the data presented in Table 1 thattransgenic plants are obtained which produce tubers having an alteredspecific gravity as compared to the tubers from control plants.

Statistical analysis is conducted on the specific gravity measurementsof tubers from the 5 replicates of one of the transformation events ascompared to the - specific gravity measurements of tubers from twocontrol events. The event analyzed is 1457-4 which has an averagespecific gravity of 1.060. The specific gravity measurements of tubersfrom the individual replicates that are used to calculate the averagefor this event are 1.059, 1.057, 1.067, 1.066, and 1.053. The specificgravity measurements for replicates of control tubers are as follows. Tx(ave. 1.079): 1.076, 1.082, 1.073, 1.083, and 1.079. Rg (ave. 1.081):1.076, 1.087, 1.083, 1.082, and 1.077. These measurements are convertedto percent solids as described above and the percent solids values areused for statistical analysis as follows.

A comparison of sample means is conducted on the percent solids valuescalculated for the three events, 1457-4, Tx and Rx, by calculating the tvalue (Student's t) and determining statistical difference based on astandard table of values for t. (See, for example, Steel and Torrie(1980) Principles and Procedures of Statistics: A Biometrical Approach(McGraw-Hill pub.) Chapter 5 and Table A.3). These analyses indicate asignificant difference between the average specific gravity measurementsof transgenic tubers as compared to control tubers at a confidence levelof greater than 99%. The average specific gravity measurements of thetwo control groups are not significantly different.

Further analysis may be conducted on tubers from selected pCGN1457 andpCGN1457B transformed plants and from non-transformed controls (RB-43)to determine starch content, amylose percentages and to elucidate chainlength distribution in the amylopectin component of the starch. Starchgranules are isolated as described by Boyer et al. (1976) CerealChemistry 53:327-337) and starch content estimated on a weight basis(starch wt/fresh wt). Amylose percentages are determined bygel-filtration analysis (Boyer et al. (1985) Starch/Starke 37:73-79).Chain length distribution patterns are determined by HPLC analysis asdescribed by Sanders et al. (1990) Cereal Chemistry 67:594-602).Amylopectins are characterized by the ratios (on a weight basis) of lowmolecular weight chains to high molecular weight chains as described byHizukuri (Carbohydrate Research (1985) 141:295-306). Results of theseanalyses are presented in Table 2.

                  TABLE 2                                                         ______________________________________                                        Analyses of Trangenic Potato Tuber Starch                                                                    %     %     Low                                                               High  Low   M.W./                                      Spec.   %       %      M.W.  M.W.  High                               Construct                                                                             Gravity Starch  Amylose                                                                              Chains                                                                              Chains                                                                              M.W.                               ______________________________________                                        RB-43   1.081   17.1    23     33    66    2.0                                1457-4  1.060   11.0    12     20    80    4.0                                1457-17 1.069   14.6    24     28    72    2.6                                1457-18 1.060   11.8    8      15    85    5.7                                RB-43   1.077   17.2    27                                                    1457B-15                                                                              1.053   9.0     9      15    85    5.7                                1457B-17                                                                              1.053   12.5    19     26    84    3.2                                ______________________________________                                    

The data presented in Table 2 indicate that tubers, from transgenicplants which have an altered specific gravity, also have altered starch.In particular, the percentage of amylose in the transgenic potato tubersis decreased. In addition, the amylopectin portion of the starch fromtransgenic potato tubers has more low molecular weight chains and lesshigh molecular weight chains than wild type potato tuber amylopectin,thus indicating that the amylopectin from transgenic tubers has morebranch points.

It is evident from the above results, that plant cells and plants can beproduced which have improved properties or may produce a desiredproduct. In accordance with the subject invention, it is now seen thatglycogen biosynthesis enzyme sequences may be introduced into a planthost cell and be used to express such enzyme or enzymes or to modifynative starch precursors. Moreover, it is seen that such enzymesdemonstrate biological activity on plant starch precursors resulting ina demonstrable phenotype in planta, namely altered specific gravity. Inaddition, the activity of glycogen biosynthetic enzymes in plants hasbeen shown to result in starch having altered properties, in particularaltered ratios of amylose/amylopectin and altered distribution of lowmolecular weight chain lengths to high molecular weight chain lengths inthe amylopectin fraction. In this manner, plants, including plant cellsand plant parts, having modified starch properties may be obtained,wherein the modified starch has unique and desirous properties.

In order to demonstrate the use of starch degradation product enzymes toproduce CGT compounds in accordance with the present invention, thefollowing examples demonstrate the creation of CGT structural geneconstructs and the transfer of such constructs into plant expressionsystems.

EXAMPLE 7 Cloning the CGT Coding Region

This example describes the isolation of the coding region for acyclodextrin glycosyltransferase (CGT) gene from Klebsiella pneumoneaeand the engineering of the coding region for subsequent cloning.

Total genomic DNA is prepared from Klebsiella pneumoneae M5A1 (Binder etal., Gene (1986) 47:269-277) by growing a 5 ml culture in ECLB(Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbon, N.Y. (1982)) overnight at 37° C. The bacteria arepelleted by centrifugation for 10 minutes at 4500×g, the supernatant isdiscarded, and the pellet is resuspended in 2.5 ml of 10 mM Tris, 1 mMEDTA buffer. To this suspension is added 500 μl of a 5 mg/ml Pronase®protease (Calbiochem Brand Biochemials; La Jolla, Calif.) solution and 2ml of 2% lauryl sulfate, sodium salt (Sigma; St. Louis, Mo.), withgentle mixing and the suspension is incubated at 37° C. for 50 minutes.A clear solution indicates that the bacteria have lysed. The solution isthen extracted with 5 ml phenol, then 5 ml phenol:chloroform:isoamylalcohol (25:24:1), followed by 5ml chloroform. Nucelic acids areprecipitated from the aqueous phase with 1/10 volume of 3M sodiumacetate and two volumes of 100% ethanol, and the tube is incubated atroom temperature for 1 hour. Nucleic acids are removed from solution andresuspended in 1 ml water. A second ethanol precipitation is performedand the nucleic acids are resuspended in 200 μl of 10 mM Tris, 1 mM EDTAbuffer.

Oligonucleotide probes flanking the 2 kb cyclodextringlycosyltransferase (EC 2.4.1.19) gene of K. pneumoneae (Bender, H.,Arch. Microbiol. (1977) 111:271-282) and containing restriction sitesfor BamHI and SalI are synthesized on an Applied Biosystems 380A DNAsynthesizer (Foster City, Calif.) in accordance with the manufacturer'sinstructions. Specifically the probes are: ##STR2##

The nucleic acid preparation of K. pneumoneae is treated with RNAse andthe DNA is used as a template in a polymerase chain reaction (PCR) withstr3 and str4 as primers. A Perkin-Elmer/Cetus (Norwalk, Conn.) thermalcycler is used with the manufacturer's reagents and in accordance withthe manufacturer's instructions. The reaction mixture contains 41.5 μ.H2O, 10 μl 10× Reaction buffer, 16 μl dNTP's (1.25 mM dCTP, dATP, dGTP &dTTP!, 5 μl str3 (20 mM), 5 μl str4 (20 mM), 22 μl total K. pneumoneaeDNA (0.05 μg/μl), and 0.5 μl Tag polymerase. The reaction is performedfor 15 cycles with melting (denaturation) for 1 minute at 94° C.,annealing (hybridization) for 2 minutes at 37° C. and chain elongationfor 3 minutes at 72° C. The reaction is then performed for an additional10 cycles with melting for 1 minute at 94° C., annealing or 2 minutes at37° C. and chain elongation at 72° C. for 3 minutes 15 seconds initiallyand increasing the time by 15 seconds each cycle so that the last cycleis 5 minutes 45 seconds.

The resulting PCR product fragments (-2 kb) are digested with SalI andBamHI and ligated into a SalI and BamHI digest of pCGN65α3X (see below).Transformed E. coli DH5α cells (BRL; Gaithersburg, Md.) containingpCGN65α3X are screened on 1% starch plates (ECLB+1% starch) by floodingwith I₂ /KI and evaluating for clearing of starch from around the edgeof the colony.

Clone 1 exhibited a good zone of clearing and is digested with SphI andSalI, ligated into SphI- and SalI-digested pUC19 (Norrander et al., Gene(1983) 26:101-106) and Yanisch-Perron et al., Gene (1985) 33:103-119),yielding the plasmid pCGT2 (-4.5kb). Sequence analysis of pCGT2 (FIG. 4and SEQ ID NOS: 15) showed six single base changes randomly distributedthroughout the CGT gene (99.7% homology) which resulted in three aminoacid changes (FIG. 4B and SEQ ID NOS: 16, 18 and 20). Plasmid pCGT2 isdigested with SphI, treated with the Klenow fragment of DNA polymerase I(Klenow fragment) to generate blunt ends and to ligate in a BglIIlinker. The resulting plasmid, pCGT4, is sequenced using the Sequenase®DNA sequencing kit (U.S. Biochemical; Cleveland, Ohio) in accordancewith the manufacturer's instructions to confirm the correct readingframe: ##STR3##

Construction of pCGN65α3X

Plasmid pUC18 (Yanisch-Perron et al., (1985) supra) is digested withHaeII to release the lacZ' fragment, treated with Klenow fragment tocreate blunt ends, and the lacZ'-containing fragment is ligated intopCGN565Rβ-H+X (see below), which has been digested with AccI and SphI,and treated with Klenow fragment, resulting in plasmid pCGN565RBα3X. InpCGN565RBα3X, the lac promoter is distal to the T-DNA right border. Bothclones are positive for lacZ' expression when plated on an appropriatehost. Each clone contains coordinates 13990-14273 of the T-DNA rightborder fragment (Barker et al., Plant mol. Biol. (1983) 2:335-350),having deleted the AccI-SphI fragment (coordinates 13800-13989). The 728bp BglII-XhoI fragment of pCGN565RBα3X, containing the T-DNA rightborder piece and the lacZ' gene, is cloned into BglII- and XhoI-digestedpCGN65ΔKX-S+X to replace the BglII-XhoI right border fragment ofpCGN65ΔKX-S+X and create pCGN65α3X. The construction of pCGN65α3X isdescribed in detail in co-pending U.S. application Ser. No. 07/382,176,filed Jul. 19, 1989.

Construction of pCGN565Rβ-H+X

Plasmid pCGN451 includes an octopine cassette which containsapproximately 1556 bp of the 5' non-coding region fused, via an EcoRIlinker, to the 3' non-coding region of the octopine synthase gene ofpTiA6. The pTi coordinates are 11,207 to 12,823 for the 3' region and13,643 to 15,208 for the 5' region (Barker et al., (1983) supra).Plasmid pCGN451 is digested with HpaI and ligated in the presence ofsynthetic SphI linker DNA to generate pCGN55. The XhoI-SphI fragment ofpCGN55 (coordinates 13800-15208, including the right border ofAgrobacterium tumefaciens T-DNA (Barker et al., Gene (1977) 2:95-113) iscloned into SalI- and SphI-digested pUC19 (Yanisch-Perron et al., (1985)supra) to create pCGN60. The 1.4kb HindIII-BamHI fragment of pCGN60 iscloned into HindIII- and BamHI-digested with pSP64 (Promega, Inc.) togenerate pCGN1039. Plasmid pCGN1039 is digested with SmaI and NruI(deleting coordinates 14273-15208 (Barker et al., (1977) supra) andligated in the presence of synthetic BglII linker DNA to createpCGN1039ΔNS. The 0.47kb EcoRI-HindIII fragment of pCGN1039ΔNS is clonedinto EcoRI- and HindIII-digested pCGN565 to create pCGN565RB. TheHindIII site of pCGN565RB is replaced with an XhoI site by HindIIIdigestion, treatment with Klenow fragment, and ligation in the presenceof synthetic XhoI linker DNA to create pCGN565Rβ-H+X.

EXAMPLE 8 Plastid Translocating Sequences

This example describes the preparation of DNA sequences encoding transitpeptides for use in the delivery of a CGT gene to starch-containingorganelles.

Construction of SSU+aroA Transit Peptide

Plasmid pCGN1132 contains a 35S promoter-ribulosebisphosphatecarboxylase small subunit (5'-35S-SSU) leader plus 48 bp of mature smallsubunit (SSU) protein from pea aroA sequence (the gene locus whichencodes 5-enolpyruvyl-3-phosphoshikimate synthetase (EC 2.5.1.19)). Itis prepared from pCGN1096, a plasmid containing a hybrid SSU proteingene, which carries DNA encoding mature SSU protein from pea, and SstIand EcoRI sites 3' of the coding region (used in the preparation ofpCGN1115, a plasmid having a 5'-35S-SSU+48-aroA-tml-3' sequence, andpCGN1129, a plasmid having a 35S promoter in a chloramphenicolresistance gene (Cam^(r)) backbone).

Construction of pCGN1096

The aroA moiety of pCGN1077 is removed by digestion with SphI and SalI.In its place is cloned the region coding for the mature pea SSU protein,as an SphI-PstI fragment, which is then excised with SphI and SalI. Theresulting plasmid, pCGN1094, codes for a hybrid SSU protein having thetransit peptide of the soybean clone, and the mature portion of the peaclone and carrier SstI and EcoRI sites 3' of the coding region. TheHindIII to BamHI region of transposon Tn6 (Jorgensen et al., Mol. Gen.Genet. (1979) 177:65) encoding the kanamycin resistance gene (Kan^(r))is cloned into the same sites of pBR322 (Bolivar et al., Gene (1977)2:95-133) generating pDS7. The BglII site 3' of the Kan^(r) gene isdigested and filled in with the large fragment of E. coli DNA polymerase1 and deoxy-nucleotides triphosphate. An SstI linker is ligated into theblunted site, generating plasmid pCGN1093. Plasmid pPMG34.3 is digestedwith SalI, the site filled in as above and EcoRI linkers are ligatedinto the site resulting in plasmid pCGN1092. The latter plasmid isdigested with SstI and SmaI and the Kan^(r) gene excised from pCGN1093with SstI and SmaI is ligated in, generating pCGN1095. The Kan^(r) andaroA genes are excised as a piece from pCGN1095 by digestion with SstIand EcoRI and inserted into the SstI and EcoRI sites of pCGN1094,producing pCGN1096. Summarizing, pCGN1096 contains (5'->3') thefollowing pertinent features: The SSU gene--a polylinker coding forPstI, SalI, SstI, and KpnI--the Kan^(r) gene--SmaI and BamHI restrictionsites--the aroA gene without the original ATG start codon. Theconstruction of pCGN1096 is also described in detail in co-pending U.S.application Ser. No. 06/097,498, filed Sep. 16, 1987.

Plasmid pCGN1096 is digested to completion with SalI and then digestedwith exonuclease Bal31 (BRL; Gaithersburg, Md.) for 10 minutes, thusdeleting a portion of the mature SSU gene. The resulting plasmid is thendigested with SmaI to eliminate the Kan^(r) gene and provide blunt ends,recircularized with T4 DNA ligase and transformed into E. coli LC3(Comai et al., Science (1983) 221:370-371), an aroA mutant. DNA isolatedfrom aroA⁺ and Kan^(r) colonies is digested with BamHI and SphI andligated with BamHI⁻ and SphI-digested Ml3mp18 (Norrander et al., Gene(1983) 26:101-106 and Yanisch-Perron et al., Gene (1985) 33:103-119) DNAfor sequencing. Clone 7 has 48 bp of the mature SSU gene remaining (FIG.1), and the 3' end consists of phe-glu-thr-leu-ser. Clone 7 istransformed into E. coli strain 71-18 (Yanisch-Perron et al. (1985)supra) and DNA isolated from transformants is digested with SphI andClaI to remove the 0.65kb fragment containing the 48 bp of matureprotein and the 5' end of the aroA gene. Plasmid pCGN1106 (Comai et al.,J. Biol. Chem. (1988) 263:15104-15109) is also digested with SphI andClaI and the 6.8 kb isolated vector fragment is ligated with the 0.65 kbfragment of clone 7 to yield pCGN1115 (5'-35S-SSU+48-aroA-tml-3').

The 7.2 kb plasmid pCGN1180 (35S-SSU+70-aroA-ocs3') (Comai et al. (1988)supra) and the 25.6 kb plasmid pCGN594 (Houck, et al., Frontiers inApplied Microbiology (1990) 4:1-17) (Lβ-Gent^(r) -ocs5'-Kan^(r)-ocs3'-RB) (construction of pCGN594 is described in co-pending U.S.application Ser. No. 07/382,802, filed Jul. 19, 1989) are digested withHindIII and ligated together to yield the 32.8 kb plasmid pCGN1109(Lβ-Gent^(r) -35S-SSU+70-aroA-ocs3-ocs5'-Kan^(r) r-ocs3' -RB).

Plasmid pCGN11O9 is digested with EcoRI to delete an internal 9.1 kbfragment containing the SSU leader plus 70 bp of the mature SSU gene,the aroA gene and its ocs3' terminator, the Amp^(r) backbone frompCGN1180 and ocs5l-Kan^(r) -ocs3l from pCGN594. The EcoRI digest ofpCGN1109 is then treated with Klenow fragment to blunt the ends, and aXhoI linker (dCCTCGAGG) (New England Biolabs Inc.; Beverly, MA) isligated in, yielding pCGN1125 (Lβ-35S-RB).

Plasmid pCGN1125 is digested with HindIII and BglII to delete the 0.72kb fragment of the 35S promoter. This digest is ligated with HindIII-and BamHI-digested Cam^(r) vector, pCGN786. Plasmid pCGN786 is achloramphenicol resistant pUC based vector formed by insertion of asynthetic linker containing restriction digest sites EcoRI, SalI, BglII,PstI, XhoI, BamHI, and HindIII into pCGN566 (pCGN566 contains theEcoRI-HindIII linker of pUC18 inserted into the EcoKI-HindIII sites ofpUC13-cm (K. Buckley (1985) Ph.D. thesis, University of California atSan Diego). The resulting 3.22kb plasmid, pCGN1128, contains the 35Spromoter with a 3' multilinker in a Cam^(r) backbone.

Plasmid pCGN1128 is digested with HindIII, treated with Klenow fragmentto blunt the ends and ligated with BglII linkers to yield pCGN1129, thuschanging the HindIII site located 5' to the 35S promoter into a BglIIsite. .

Plasmid pCGN1115 is digested with SalI to removed a 1.6 kb fragmentcontaining the SSU leader plus 48 bp of the mature SSU gene and the aroAgene. An XhoI digest of pCGN1129 opened the plasmid 3' to the 35Spromoter. Ligation of these two digests yielded the 4.8 kb plasmidpCGN1132, containing 5'-35S-SSU leader plus 48 bp of mature SSU-aroA.Plasmid pCGN1132 is digested with EcoRI, treated with Klenow fragment toform blunt ends, and ligated with SacI linkers (d(CGAGCTCG) New EnglandBiolabs Inc.; Beverly, Mass.) to yield pCGN1132S, thus changing theEcoRI site 3' to the aroA gene to a SacI site.

Transit Peptide+Cyclodextrin Glycosyltransferase Gene

Plasmid pCGT4 (See Example 7) and pCGN1132S are digested with BamHI andSalI and ligated together. The resulting plasmid pCGT5 contains5'-35S-SSU+48-CGT-3'.

EXAMPLE 9 Cloning of Patatin Regulatory Regions and Preparation ofPatatin-5'-nos-3' Expression Cassettes

This example describes the cloning of patatin-5' regulatory regions fromtwo potato varieties and the preparation of patatin-5'-nos-3' expressioncassettes pCGN2143 and pCGN2144. Also provided is the cloning ofpatatin-3' regulatory regions and the preparation ofpatatin-5'-patatin-3' expression cassettes pCGN2173 and pCGN2174.

Genomic DNA is isolated from leaves of Solanum tuberosum var. RussettBurbank and var. Kennebec as described in Dellaporta et al., Plant Mol.Biol. Reporter (1983) 1(4):19-21, with the following modifications:Approximately 9 g fresh weight of leaf tissue is ground, a polytrongrinding is not performed and in the final step the DNA is dissolved in300 μl of 10 mM Tris, 1 mM EDTA, pH 8. A synthetic oligonucleotide,pat1, containing digestion sites for NheI, PstI and XhoI with 24 bp ofhomology of the 5'-region of a 701 bp fragment (coordinates 1611 to2312) 5' to a class I patatin gene, isolated from Solanum tuberosum var.Maris Piper (Bevan et al., NAR (1986) 14:4625-4638) is synthesized(Applied BioSystems 380A DNA synthesizer): pat 1: ##STR4##

A second synthetic oligonucleotide, pat2, containing digestion sites forBamHI and SpeI with 25 bp of homology to the 3' region of the 701 bppiece is also synthesized: pat2: ##STR5##

Using the genomic potato DNA as a template, and pat1 and pat2 asprimers, a polymerase chain reaction (PCR) is performed in aPerkin-Elmer/Cetus thermal cycler with the manufacturer's reagents andin accordance with the manufacturer's instructions. The reactioncontains 62.5 μH₂ O, 10 μl 10× Reaction buffer, 16 μl dNTP's (1.25 mMdCTP, dATP, dGTP & dTTP!, 5 μl pat1 (20 mM), 5 μl pat2 (20 mM), 1 μlpotato genomic DNA (3 μg/μl), 0.5 μl Taq polymerase. The PCR isperformed for 25 cycles with melting for 1 minute at 94° C., annealingfor 2 minutes at 37° C. and chain elongation for 3 minutes at -72° C.The resulting PCR product fragments (approximately 700 bp) are digestedwith NheI and BamHI. Plasmid pCGN1586N (5'-D35S-TMVΩ''-nos'3'; pCGN1586(described below) having a NheI site 5' to the 35S region) is digestedwith NheI and BamHI to delete the D35S-Ω' fragment. Ligation ofNheI-BamHI digested pCGN1586N, which contains the nos-3' region, and thePCR fragments yield a patatin-5'-nos-3' cassette with SpeI, BamHI, SalIand SstI restriction sites between the 5' and 3' regions for insertionof a DNA sequence of interest.

The 5' regions of two clones, designated pCGN2143 and pCGN2144, aresequenced. Plasmid pCGN2143 has a Kennebec patatin-5' region that is 702bp in length and 99.7% homologous to the native sequence (as reported byBevan (1986) supra) (FIG. 2). The 5' region of pCGN2144, from RussetBurbank, is 636bp in length, containing a 71 bp deletion from coordinate1971 to coordinate 2040. The remainder of the Russet Burbank clone is97.0% homologous to the native sequence (as reported by Bevan (1986)supra) (FIG. 3).

A synthetic oligonucleotide, pat3S, with 24bp of homology to the 5'region of a 804 bp region 3' to a class I patatin gene (Bevan 5000 to5804):

pat3S: ##STR6## is synthesized. This oligonucleotide contained arestriction enzyme site for SstI. A second oligonucleotide, pat4, with24 bp of homology to the 3' region of the 804 bp region is alsosynthesized:

pat4: ##STR7## It contains digestion sites for the enzymes NheI, XhoIand PstI.

Using Russet Burbank genomic potato DNA as a template, a polymerasechain reaction (PCR) as described above is performed for 25 cycles withmelting for 1 minute at 94° C., annealing for 2 minutes at 42° C. andchain elongation for 3 minutes at 72° C. A Perkin-Elmer/Cetus thermalcycler is used with the manufacturer's reagents and in accordance withthe manufacturer's instructions. Specifically, the reaction contained53.5 μl H₂ O, 10 μl 10× reaction buffer, 16 μl dNTP's 1.25mM dCTP, dATP,dGTP & dTTP!, 5 μl pat3S (20 mM), 5 μl pat4 (20 mM), 10 μl genomicpotato DNA (3 μg/μl), 0.5 μl Tag polymerase. The resulting approximately800 bp PCR product fragments are digested with NheI and SstI and ligatedinto pCGN1586N (see below). Sequencing of one clone, designatedpCGN2159, showed that the 3' fragment is 823 bp in length and 93.6%homologous to Bevan's reported sequence (Bevan (1986) supra).

Cloning of the patatin cassettes PCGN2173 and PCGN2174

A patatin cassette consisting of the 5' patatin region from Kennebec and3' patatin region from Russet Burbank, identified as pCGN2173, isconstructed by a three way ligation of the following fragments: The NheIto SstI Kennebec 5' patatin fragment of pCGN2143 (see above), the SstIto NheI Russet Burbank 3' patatin fragment of pCGN2159 and the NheI toNheI pUC backbone of pCGN1599.

A second patatin cassette, identified as pCGN2174, is constructed by athree way ligation of the NheI to SstI Russet Burbank 5' patatinfragment of pCGN2144 (see above), the SstI to NheI Russet Burbank 3'patatin fragment of pCGN2159 and the NheI to NheI pUC backbone ofpCGN1599.

Construction of PCGN1586/l586N

Plasmid pCGN2113 (6.1 kb) contains a double-35S promoter (D35S) and thetml-3' region with multiple cloning sites between them, contained in apUC-derived plasmid backbone bearing an ampicillin resistance gene(Amp^(r)). The promoter/tml cassette is bordered by multiple restrictionsites for easy removal. Plasmid pCGN2113 is digested with EcoRI andSacI, deleting the 2.2 kb tml-3' region. Plasmid pBI221.1 (Jefferson, R.A., Plant Mol. Biol. Reporter (1987) 5:387-405) is digested with EcoRIand SacI to delete the 0.3 kb nos-3' region. The digested pCGN2113 andpBI221.1 DNAs are ligated together, and the resultant 4.2 kb recombinantplasmid with the tml-3' of pCGN2113 replaced by nos-3' is designatedpCGN1575 (5'-D35S-nos-3').

Plasmid pCGN1575 is digested with SphI and XbaI, blunt ends generated bytreatment with Klenow fragment, and the ends are ligated together. Inthe resulting plasmid, pCGN1577, the Sph, PstI, SalI and XbaI sites 5'of the D35S promoter are eliminated.

Plasmid pCGN1577 is digested with EcoRI, the sticky ends blunted bytreatment with Klenow fragment, and synthetic BglII linkers(d(pCAGATCTG) New England Biolabs Inc.; Beverly, Mass.) are ligated in.A total of three BglII linkers are ligated into the EcoRI site creatingtwo PstI sites. The resulting plasmid, termed pCGN1579 (D35S-nos-3'),has a 3' polylinker consisting of 5'-EcoRI, BglII, PstI, BglII, PstI,BglII, EcoRI-3'.

A tobacco Mosaic Virus omega' (TMVΩ') region (Gallie et al., NAR (1987)15(21) :8693-8711) with BglII, NcoI, BamHI, SalI and SacI restrictionsites: ##STR8## is synthesized on a Applied Biosystems® 380A DNAsynthesizer and digested with BglII and SacI. Plasmid pCGN1577 isdigested with BamHI and SacI and the synthetic TMVΩ' is ligated inbetween the 5'-D35S and nos-3' regions. The resulting plasmid isdesignated pCGN1586 (5'-D35S-TMVΩ'-nos'3'). Plasmid pCGN1586N is made bydigesting pCGN1586 with HindIII and filling in the 5' overhang withKlenow fragment, thus forming a NheI site 5' to the D35S region.

EXAMPLE 10 Preparation of Patatin-5'-CGT-Nos-3' Binary Vectors

This example describes the construction of binary vectors containing:(1) the patatin-5' region from either Solanum tuberosum var. Kennebec orvar. Russet Burbank, (2) DNA encoding a transit peptide from soybeanRuBisCo SSU protein, (3) 48 bp of DNA encoding 16 amino acids of matureRuBisCo SSU protein from pea, (4) the CGT coding region from Klebsiellapneumoneae, and (5) the nos-3' region.

Plasmid pCGN2143 prepared as described in Example 9 is digested withSpeI and SstI, opening the plasmid between the patatin-5' region andnos-3' region. Plasmid pCGT5 (see Example 8) is digested with XbaI andSstI and ligated with pCGN2143 to yield pCGN2151. Plasmid pCGN2151consists of 5'-Kennebec patatin-SSU+48-CGT-nos3'. Plasmid pCGN2151 isdigested with PstI and ligated with PstI-digested pCGN1558 (see below).This yields the binary vectors pCGN2160a and pCGN2160b.

In pCGN2160a, the 5'-patatin-SSU+48bp-CGT-nos 3' is inserted intopCGN1558 such that it transcribes in the opposite direction as the35S-Kan^(r) -tml gene. In pCGN2160b, the 5'-patatin-SSU+48bp-CGT-nos-3'is inserted into pCGN1558 such that it transcribes in the same directionas the 35S-Kan^(r) -tml gene.

Plasmid pCGN2144 is digested with SpeI and SstI, opening the plasmidbetween the patatin-5¹ and nos-3' regions. Plasmid pCGT5 is digestedwith XbaI and SstI and ligated with pCGN2144 to yield pCGN2152. PlasmidpCGN2152 consists of 5'-Russet Burbank patatin-SSU+48-CGT-nos3'. PlasmidpCGN2152 is digested with PstI and ligated with pCGN1558 (see below)digested with PstI. This yields the binary vectors pCGN2161a andpCGN2161b. In pCGN2161a, the 5'-patatin-SSU+48 bp-CGT-nos3' is insertedinto pCGN1558 such that it transcribes in the opposite direction as the35S-Kan^(r) -tml gene. In pCGN2161b, the 5'-patatin-SSU+48bp-CGT-nos-3'is inserted into pCGN1558 such that it transcribes in the same directionas the 35S-Kan^(r) -tml gene.

Construction of pCCGN1558

Plasmid pCGN1558 (McBride and Summerfelt, Plant Mol. Biol. (1990) 14(27):269-276) is a binary plant transformation vector containing the leftand right T-DNA borders of Agrobacterium tumefaciens octopine Ti-plasmidpTiA6 (Currier and Nester, J. Bact. (1976) 126:157-165), the gentamicinresistance gene (Gen^(r)) of pPH1JI (Hirsch and Beringer, Plasmid (1984)12:139-141) an Agrobacterium rhizogenes Ri plasmid origin of replicationfrom pLJbB11 (Jouanin et al., Mol. Gen. Genet. (1985) 201:370-374), a35S promoter-Kan^(r) -tml-31 region capable of conferring kanamycinresistance to transformed plants, a ColE1 origin of replication frompBR322 (Bolivar et al. (1977) supra) and a lacZ' screenable marker genefrom pUC18 (Yanish-Perron et al. (1985) supra). The construction ofpCGN1558 is described in co-pending U.S. application Ser. No.07/494,722, filed Mar. 16, 1990.

EXAMPLE 11 Preparation of Transgenic Plants

This example describes the transformation of Agrobacterium tumefacienswith a CGT gene DNA construct in accordance with the present inventionand the cocultivation of such A. tumefaciens with plant cells totransform host cells and enable the resultant plants to producecyclodextrins.

Transformation of Agrobacterium tumefaciens

Cells of Agrobacterium tumefaciens strain 2760 (also known as LBA4404,Hoekema et al., Nature (1983) 303:179-180) are transformed with binaryvectors, such as pCGN2160a, pCGN2160b, pCGN2161a and pCGN2161b (asdescribed in Example 10) using the method of Holsters et al. (Mol. Gen.Genet. (1978) 163:181-187). The transformed A. tumefaciens are then usedin the cocultivation of plants, in order to transfer the CGT constructinto an expression system.

The Agrobacterium are grown in AB medium (per liter: 6 g K₂ HPO₄, 2.3 gNaH₂ PO₄.H₂ O, 2 g NH₄ Cl, 3 g KCl, 5 g glucose, 2.5 mg FeSO₄, 246 mgMgSO₄, 14.7 mg CaCl₂, 15 g agar) plus 100 μg/L gentamicin sulfate and100 μg/L streptomycin sulfate for 4-5 days. Single colonies areinoculated into 10 ml of MG/L broth (per liter: 5 g mannitol, 1 gL-Glutamic acid or 1.15 g sodium glutamate, 0.5g KH₂ PO₄, 0.10 g NaCl,0.10 g MgSO₄ 0.7H₂ O, 1 μg biotin, 5 g tryptone, 2.5 g yeast extract;adjust pH to 7.0) and are incubated overnight in a shaker at 30° C. and180 rpm. Before cocultivation, the Agrobacterium culture is centrifugedat 12,000×g for 10 minutes and resuspended in 20 ml MS medium(#510-1118, Gibco; Grand Island, N.Y.).

Cocultivation with Potato Cells

Feeder plates are prepared by pipetting 0.5 ml of a tobacco suspensionculture (˜10⁶ cells/ml) onto 0.8% agar co-cultivation medium containingMS salts (#510-117, Gibco; Grand Island, N.Y.), 1.0 mg/L thiamine-HCl,0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine-HCl, 30 g/L sucrose, 5 μMzeatin riboside, 3 μM 3-indoleacetyl-DL-aspartic acid, pH 5.9. Thefeeder plates are prepared one day in advance and incubated at 250C. Asterile 3 mm filter paper disk is placed on top of the tobacco cellsafter they have grown for one day.

Tubers of Solanum tuberosum var. Russet Burbank and var. Kennebecbetween the age of 1 and 6 months post-harvest are peeled and washed indistilled water. All subsequent steps are carried out in a flow hoodusing sterile techniques. For surface sterilization, tubers are immersedin a solution of 10% commercial bleach (sodium hypochlorite) with 2drops of Ivory® liquid soap per 100 ml for 10 minutes. Tubers are rinsedsix times in sterile distilled water and kept immersed in sterile liquidMS medium (#1118, Gibco; Grand Island; N.Y.) to prevent browning.

Tuber discs (1-2mm thick) are prepared by cutting columns of potatotuber with a 1 cm cork borer and slicing the columns to the desiredthickness. Discs are placed into the liquid MS medium culture of thetransformed A. tumefaciens containing the binary vector of interest(1×10⁷ -1×10⁸ bacteria/ml) until thoroughly wetted. Excess bacteria areremoved by blotting discs on sterile paper towels. The discs areco-cultivated with the bacteria for 48 hours on the feeder plates andthen transferred to regeneration medium (co-cultivation medium plus 500mg/L carbenicillin and 100 mg/L kanamycin). In 3 to 4 weeks, shootsdevelop from the discs.

When shoots are approximately 1 cm, they are excised and transferred toa 0.8% agar rooting medium containing MS salts, 1.0 mg/L thiamine-HCl,0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine-HCl, 30 g/L sucrose, 200mg/L carbenicillin and 100-200 mg/L kanamycin at pH 5.9. Plants arerooted two times with at least one rooting taking place on rootingmedium with the higher level of kanamycin (200 mg/L). Plants whichrooted twice are then confirmed as transformed by performing the NPTIIblot activity assays (Radke, S. E. et al., Theo. Appl. Genet. (1988)75:685-694). Plants which are not positive for NPII activity arediscarded.

Northern Blot Analysis of Transformed Plants

Total RNA is isolated from 5 g of tuber tissue (as described by Logemanet al., Anal. Biochem. (1987) 163:16-20). Poly-(A)+RNA is purified overoligo(dT) cellulose (as described by Maniatis et al. (1982) supra). RNAdenaturing gels are run and blotted (as described by Facciotti et al.,Bio/Technology (1985) 3:241-246). Equivalent amounts of poly-(A)+RNA arerun in each lane. A 1.9 kb BamHI fragment of pCGT4 containing the CGTgene is used as a probe in the hybridization. The fragment may beisolated from an agarose gel using the Gene Clean® Kit (Bio 101, Inc.;La Jolla, Calif.) in accordance with the manufacturer's instructions.Nick-translation and hybridization are performed (as described byShewmaker et al., Virology (1985) 140:281-288 except that washes are at55° C). The washed blot is autoradiographed on Kodak® X-OMat AR X-rayfilm (Rochester, N.Y.) at -70° C.

An autoradiogram of Russet Burbank potatoes each transformed with one ofpCGN2160a, pCGN2161a or pCGN2161b shows bands in each of thetransformant sample lanes. The bands are 2.3 kb in size, correspondingto the size of CGT message RNA. There is no band present in the lanecontaining RNA from the untransformed control.

EXAMPLE 12 Recovery of Cyclodextrin From Plants

In this example, the recovery and detection of cyclodextrin intransgenic potato tubers is described.

Rooted plants transformed as described in Example 11 are transplantedfrom rooting medium to a growth chamber (21° C., 16 hour photoperiodwith 250-300 μE/m² /sec light intensity) in soil prepared as follows:For about 340 gallons, combine 800 lb 20/30 sand (approximately 14 cubicfeet), 16 cubic feet Fisons° Canadian Peat Moss, 16 cubic feet #3vermiculite, and approximately 4.5 lb hydrated lime in a Gleason® mixer.The soil is steamed in the mixer for two hours; the mixer mixes forabout 15 seconds at interval of fifteen minutes over a period of onehour to ensure even heating throughout the soil. During and after theprocess of steaming, the soil reaches temperatures of at least 180° F.for one hour. The soil then sits in the mixer until the next day. Atthat time, hydrated lime is added, if necessary, to adjust the pH torange between 6.30 and 6.80.

The relative humidity of the growth chamber is maintained at 70-90% for2-4 days, after which the humidity is maintained at 40-60%. When plantsare well established in the soil, at approximately two weeks, they aretransplanted into the greenhouse. Plants are grown in 6.5 inch pots in asoil mix of peat:perlite:vermiculite (11:1:9) at an average temperatureof 24° C. day/12° C. night. Day length is approximately 12 hours andlight intensity levels varied from approximately 600 to 1000 μE/m² /sec.

Tubers are harvested from plants 14 weeks after transplant into thegreenhouse. Immediately after harvest, tubers are washed, weighed andtheir specific gravity determined. Three representative tubers from eachtransformant are peeled, rinsed in distilled water, chopped intoapproximately 0.5 cm cubes, quick frozen in liquid nitrogen, and storedat approximately -70° C until assayed. Extraction of Cyclodextrin Toprepare samples for chromatography, cubes of frozen tuber tissue areground into a powder in a coffee mill (KrupsO, Closter, N.J.). For eachplant assayed, extracts from tubers are prepared as follows: Five gramsof frozen potato powder are ground in a prechilled mortar and pestlewith 5 ml 25% ethanol and then frozen at -70° C for at least overnight.Samples are then centrifuged at 8500×g for 10 minutes, the supernatanttransferred to a clean tube, and the ethanol removed by roto-evaporationfor 1 hour.

The cyclodextrin is separated from the tissue samples in C18 SEP-PAKcolumns (Waters Chromatography Div.; Milford, Mass.), previously washedwith 5 ml of 100% methanol, followed by 5 ml of 50% methanol, followed 5ml of water prior to sample application. After the sample is applied,the cartridge is washed with 10 ml of distilled water to removecontaminants, and the cyclodextrins are removed with 0.75 ml of 100%methanol, discarding the first two drops. The sample is thenroto-evaporated to dryness, and redissolved in 20 μl of 30% methanol.Detection of Cyclodextrin Thin layer chromatography (TLC) is performedas described by Szejtli (Szejtli, J., Cyclodextrin Technology (1988) pp.20-22, Kluwer Academic Publishers, Boston). Samples are spotted onsilicagel G plates (#01011, Analtech; Newark, Del.) and dried. Thechromatogram is developed for approximately 3 hours to a height of13-15cm, with a n-butanol-ethanol-water (4:3:3) mixture. After drying,the plate is exposed to iodine vapor for 5-10 min. to visualize thechromatogram.

Positive controls of α-cyclodextrin (α-CD) and β-cyclodextrin (β-CD) arerun alongside samples from transgenic tissue, and average Rf values forfour plates are 0.39 for α-CD and 0.36 for β-CD. The α-CD band stainedlight violet, while the β-CD band stained yellow. Tuber tissue from 20transformed plants is screened for the presence of α-CD and β-CD. Tissueof tubers from eight Russet Burbank plants (RB2160α-11, RB2160b-7,RB2160b-9, RB216lα-2, RB216lb-3, RB216lb-5, RB216lb-11) produced bandswhich stained the same color as the α-CD control bands and had similarRf values. In addition to the putative α-CD bands, the tubers from twoplants . (RB2160b-7 and 2160b-9) produced bands with Rf values and colorsimilar to the β-CD control band.

In accordance with one aspect of the subject invention, cyclodextrin canbe produced by host plants by incorporation of a cyclodextringlycosyltransferase structural gene together with the appropriateregulatory sequence. In addition, DNA sequences coding for cyclodextringlycosyltransferase are provided which can be used for producingcyclodextrin, for example, in methods of the present invention. Thus,plants are grown which can produce cyclodextrin, in order to enhance theutility of the crop plants.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teaching of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 28                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       PheGluThrLeuSer                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CAGGAGATCTTATTTTTACAACAATTACCAACAACAACAAACAACAAACAACATTACAAT60                TACTATTTACAATTACACCATGGATCCGTCGACGAGCTC99                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATATAGGATCCATTAGGACTAGATAATGAAAAGAA35                                         (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AATAAGTCGACTTTTAATTAAAACGAGCCATTCGT35                                         (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CCAAGCTTGCGGATCCGCAGACGATT26                                                  (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CAGCAGGCTAGCTCGCTGCAGCATCTCGAGATTTGTCAAATCAGGCTCAAAGATC55                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ACGACGGGATCCCATACTAGTTTTGCAAATGTTCAAATTGTTTTT45                               (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CAGCAGGAGCTCGTACAAGTTGGCGAAACATTATTG36                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ACGACGGCTAGCTCGCTCGAGCATCTGCAGTGCATATAAGTTCACATTAATATG54                      (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 99 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CAGGAGATCTTATTTTTACAACAATTACCAACAACAACAAACAACAAACAACATTACAAT60                TACTATTTACAATTACACCATGGATCCGTCGACGAGCTC99                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1464 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GATCTAACAGGAGCGATAATGCAGGTTTTACATGTATGTTCAGAGATGTTCCCGCTGCTT60                AAAACCGGCGGTCTGGCTGATGTTATTGGGGCATTACCCGCAGCACAAATCGCAGACGGC120               GTTGACGCTCGCGTACTGTTGCCTGCATTTCCCGATATTCGCCGTGGCGTGACCGATGCG180               CAGGTAGTATCCCGTCGTGATACCTTCGCCGGACATATCACGCTGTTGTTCGGTCATTAC240               AACGGGGTTGGCATTTACCTGATTGACGCGCCGCATCTCTATGATCGTCCGGGAAGCCCG300               TATCACGATACCAACTTATTTGCCTATACCGACAACGTATTGCGTTTTGCGCTGCTGGGG360               TGGGTTGGGGCAGAAATGGCCAGCGGGCTTGACCCATTCTGGCGTCCTGATGTGGTGCAT420               GCGCACGACTGGCATGCAGGCCTTGCGCCTGCGTATCTGGCGGCGCGCGGGCGTCCGGCG480               AAGTCGGTGTTTACTGGGCACAACCTGGCCTATCAAGGCATGTTTTATGCACATCACATG540               AATGACATCCAATTGCCATGGTCATTCTTTAATATTCATGGGCTGGAATTCAACGGACAA600               ATCTCTTTCCTGAAGGCCGGTCTGTACTATGCCGATCACATTACGGCGGTCAGTCCAACC660               TACGCTCGCGAGATCACCGAACCGCAGTTTGCCTACGGTATGGAAGGTCTGTTGCAACAG720               CGTCACCGTGAAGGGCGTCTTTCCGGCGTACTGAACGGCGTGGACGAGAAAATCTGGAGT780               CCAGAGACGGACTTACTGTTGGCCTCGCGTTACACCCGCGATACGTTGGAAGATAAAGCG840               GAAAATAAGCGCCAGTTACAAATCGCAATGGGGCTTAAGGTTGACGATAAAGTGCCGCTT900               TTTGCAGTGGTGAGCCGTCTGACCAGCCAGAAAGGTCTCGACCTGGTGCTGGAAGCCTTA960               CCGGGTCTTCTGGAGCAGGGCGGGCAGCTGGCGCTACTCGGCGCGGGCGATCCGGTGCTG1020              CAGGAAGGTTTCCTTGCGGCGGCAGCGGAATACCCCGGTCAGGTGGGCGTTCAGATTGGC1080              TATCACGAAGCATTTTCGCATCGCATTATGGGCGGCGCGGACGTCATTCTGGTGCCCAGC1140              CGTTTTGAACCGTGCGGCTTAACGCAACTTTATGGATTGAAGTACGGTACGCTGCCGTTA1200              GTGCGGCGCACCGGTGGGCTTGCTGATACGGTTTCTGACTGTTCTCTTGAGAACCTTGCA1260              GATGGCGTCGCCAGTGGGTTTGTCTTTGAAGATAGTAATGCCTGGTCGCTGTTACGGGCT1320              ATTCGACGTGCTTTTGTACTGTGGTCCCGTCCTTCACTGTGGCGGTTTGTGCAACGTCAG1380              GCTATGGCAATGGATTTTAGCTGGCAGGTCGCGGCGAAGTCGTACCGTGAGCTTTACTAT1440              CGCTCGAAATAGTTTTCAGTCGAC1464                                                  (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 477 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      MetGlnValLeuHisValCysSerGluMetPheProLeuLeuLysThr                              151015                                                                        GlyGlyLeuAlaAspValIleGlyAlaLeuProAlaAlaGlnIleAla                              202530                                                                        AspGlyValAspAlaArgValLeuLeuProAlaPheProAspIleArg                              354045                                                                        ArgGlyValThrAspAlaGlnValValSerArgArgAspThrPheAla                              505560                                                                        GlyHisIleThrLeuLeuPheGlyHisTyrAsnGlyValGlyIleTyr                              65707580                                                                      LeuIleAspAlaProHisLeuTyrAspArgProGlySerProTyrHis                              859095                                                                        AspThrAsnLeuPheAlaTyrThrAspAsnValLeuArgPheAlaLeu                              100105110                                                                     LeuGlyTrpValGlyAlaGluMetAlaSerGlyLeuAspProPheTrp                              115120125                                                                     ArgProAspValValHisAlaHisAspTrpHisAlaGlyLeuAlaPro                              130135140                                                                     AlaTyrLeuAlaAlaArgGlyArgProAlaLysSerValPheThrGly                              145150155160                                                                  HisAsnLeuAlaTyrGlnGlyMetPheTyrAlaHisHisMetAsnAsp                              165170175                                                                     IleGlnLeuProTrpSerPhePheAsnIleHisGlyLeuGluPheAsn                              180185190                                                                     GlyGlnIleSerPheLeuLysAlaGlyLeuTyrTyrAlaAspHisIle                              195200205                                                                     ThrAlaValSerProThrTyrAlaArgGluIleThrGluProGlnPhe                              210215220                                                                     AlaTyrGlyMetGluGlyLeuLeuGlnGlnArgHisArgGluGlyArg                              225230235240                                                                  LeuSerGlyValLeuAsnGlyValAspGluLysIleTrpSerProGlu                              245250255                                                                     ThrAspLeuLeuLeuAlaSerArgTyrThrArgAspThrLeuGluAsp                              260265270                                                                     LysAlaGluAsnLysArgGlnLeuGlnIleAlaMetGlyLeuLysVal                              275280285                                                                     AspAspLysValProLeuPheAlaValValSerArgLeuThrSerGln                              290295300                                                                     LysGlyLeuAspLeuValLeuGluAlaLeuProGlyLeuLeuGluGln                              305310315320                                                                  GlyGlyGlnLeuAlaLeuLeuGlyAlaGlyAspProValLeuGlnGlu                              325330335                                                                     GlyPheLeuAlaAlaAlaAlaGluTyrProGlyGlnValGlyValGln                              340345350                                                                     IleGlyTyrHisGluAlaPheSerHisArgIleMetGlyGlyAlaAsp                              355360365                                                                     ValIleLeuValProSerArgPheGluProCysGlyLeuThrGlnLeu                              370375380                                                                     TyrGlyLeuLysTyrGlyThrLeuProLeuValArgArgThrGlyGly                              385390395400                                                                  LeuAlaAspThrValSerAspCysSerLeuGluAsnLeuAlaAspGly                              405410415                                                                     ValAlaSerGlyPheValPheGluAspSerAsnAlaTrpSerLeuLeu                              420425430                                                                     ArgAlaIleArgArgAlaPheValLeuTrpSerArgProSerLeuTrp                              435440445                                                                     ArgPheValGlnArgGlnAlaMetAlaMetAspPheSerTrpGlnVal                              450455460                                                                     AlaAlaLysSerTyrArgGluLeuTyrTyrArgSerLys                                       465470475                                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1323 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GATCTAGGAGCGATAATGGTTAGTTTAGAGAAGAACGATCACTTAATGTTG51                         MetValSerLeuGluLysAsnAspHisLeuMetLeu                                          1510                                                                          GCGCGCCAGCTGCCATTGAAATCTGTTGCCCTGATACTGGCGGGAGGA99                            AlaArgGlnLeuProLeuLysSerValAlaLeuIleLeuAlaGlyGly                              152025                                                                        CGTGGTACCCGCCTGAAGGATTTAACCAATAAGCGAGCAAAACCGGCC147                           ArgGlyThrArgLeuLysAspLeuThrAsnLysArgAlaLysProAla                              303540                                                                        GTACACTTCGGCGGTAAGTTCCGCATTATCGACTTTGCGCTGTCTAAC195                           ValHisPheGlyGlyLysPheArgIleIleAspPheAlaLeuSerAsn                              45505560                                                                      TGCATCAACTCCGGGATCCGTCGTATGGGCGTGATCACCCAGTACCAG243                           CysIleAsnSerGlyIleArgArgMetGlyValIleThrGlnTyrGln                              657075                                                                        TCCCACACTCTGGTGCAGCACATTCAGCGCGGCTGGTCATTCTTCAAT291                           SerHisThrLeuValGlnHisIleGlnArgGlyTrpSerPhePheAsn                              808590                                                                        GAAGAAATGAACGAGTTTGTCGATCTGCTGCCAGCACAGCAGAGAATG339                           GluGluMetAsnGluPheValAspLeuLeuProAlaGlnGlnArgMet                              95100105                                                                      AAAGGGGAAAACTGGTATCGCGGCACCGCAGATGCGGTCACCCAAAAC387                           LysGlyGluAsnTrpTyrArgGlyThrAlaAspAlaValThrGlnAsn                              110115120                                                                     CTCGACATTATCCGCCGTTATAAAGCGGAATACGTGGTGATCCTGGCG435                           LeuAspIleIleArgArgTyrLysAlaGluTyrValValIleLeuAla                              125130135140                                                                  GGCGACCATATCTACAAGCAAGACTACTCGCGTATGCTTATCGATCAC483                           GlyAspHisIleTyrLysGlnAspTyrSerArgMetLeuIleAspHis                              145150155                                                                     GTCGAAAAAGGCGCACGTTGCACCGTTGCTTGTATGCCAGTACCGATT531                           ValGluLysGlyAlaArgCysThrValAlaCysMetProValProIle                              160165170                                                                     GAAGAAGCCTCCGCATTTGGCGTTATGGCGGTTGATGAGAACGATAAA579                           GluGluAlaSerAlaPheGlyValMetAlaValAspGluAsnAspLys                              175180185                                                                     ATTATCGAATTCGTTGAAAAACCTGCTAACCCGCCGTCAATGCCGAAC627                           IleIleGluPheValGluLysProAlaAsnProProSerMetProAsn                              190195200                                                                     GATCCGAGCAAATCTCTGGCGAGTATGGGTATCTACGTCTTTGACGCC675                           AspProSerLysSerLeuAlaSerMetGlyIleTyrValPheAspAla                              205210215220                                                                  GACTATCTGTATGAACTGCTGGAAGAAGACGATCGCGATGAGAACTCC723                           AspTyrLeuTyrGluLeuLeuGluGluAspAspArgAspGluAsnSer                              225230235                                                                     AGCCACGACTTTGGCAAAGATTTGATTCCCAAGATCACCGAAGCCGGT771                           SerHisAspPheGlyLysAspLeuIleProLysIleThrGluAlaGly                              240245250                                                                     CTGGCCTATGCGCACCCGTTCCCGCTCTCTTGCGTACAATCCGACCCG819                           LeuAlaTyrAlaHisProPheProLeuSerCysValGlnSerAspPro                              255260265                                                                     GATGCCGAGCCGTACTGGCGCGATGTGGGTACGCTGGAAGCTTACTGG867                           AspAlaGluProTyrTrpArgAspValGlyThrLeuGluAlaTyrTrp                              270275280                                                                     AAAGCGAACCTCGATCTGGCCTCTGTGGTGCCGGAACTGGATATGTAC915                           LysAlaAsnLeuAspLeuAlaSerValValProGluLeuAspMetTyr                              285290295300                                                                  GATCGCAATTGGCCAATTCGCACCTACAATGAATCATTACCGCCAGCG963                           AspArgAsnTrpProIleArgThrTyrAsnGluSerLeuProProAla                              305310315                                                                     AAATTCGTGCAGGATCGCTCCGGTAGCCACGGGATGACCCTTAACTCA1011                          LysPheValGlnAspArgSerGlySerHisGlyMetThrLeuAsnSer                              320325330                                                                     CTGGTTTCCGACGGTTGTGTGATCTCCGGTTCGGTGGTGGTGCAGTCC1059                          LeuValSerAspGlyCysValIleSerGlySerValValValGlnSer                              335340345                                                                     GTTCTGTTCTCGCGCGTTCGCGTGAATTCATTCTGCGACATTGATTCC1107                          ValLeuPheSerArgValArgValAsnSerPheCysAspIleAspSer                              350355360                                                                     GCCGTATTGTTACCGGAAGTATGGGTAGGTCGCTCGTGCCGTCTGCGC1155                          AlaValLeuLeuProGluValTrpValGlyArgSerCysArgLeuArg                              365370375380                                                                  CGCTGCGTCATCGATCGTGCTTGTGTTATTCCGGAAGGCATGGTGATT1203                          ArgCysValIleAspArgAlaCysValIleProGluGlyMetValIle                              385390395                                                                     GGTGAAAACGCAGAGGAAGATGCACGTCGTTTCTATCGTTCAGAAGAA1251                          GlyGluAsnAlaGluGluAspAlaArgArgPheTyrArgSerGluGlu                              400405410                                                                     GGCATCGTGCTGGTAACGCGCGAAATGCTACGGAAGTTAGGGCATAAA1299                          GlyIleValLeuValThrArgGluMetLeuArgLysLeuGlyHisLys                              415420425                                                                     CAGGAGCGATAATGCAGGGTCGAC1323                                                  GlnGluArg                                                                     430                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 431 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      MetValSerLeuGluLysAsnAspHisLeuMetLeuAlaArgGlnLeu                              151015                                                                        ProLeuLysSerValAlaLeuIleLeuAlaGlyGlyArgGlyThrArg                              202530                                                                        LeuLysAspLeuThrAsnLysArgAlaLysProAlaValHisPheGly                              354045                                                                        GlyLysPheArgIleIleAspPheAlaLeuSerAsnCysIleAsnSer                              505560                                                                        GlyIleArgArgMetGlyValIleThrGlnTyrGlnSerHisThrLeu                              65707580                                                                      ValGlnHisIleGlnArgGlyTrpSerPhePheAsnGluGluMetAsn                              859095                                                                        GluPheValAspLeuLeuProAlaGlnGlnArgMetLysGlyGluAsn                              100105110                                                                     TrpTyrArgGlyThrAlaAspAlaValThrGlnAsnLeuAspIleIle                              115120125                                                                     ArgArgTyrLysAlaGluTyrValValIleLeuAlaGlyAspHisIle                              130135140                                                                     TyrLysGlnAspTyrSerArgMetLeuIleAspHisValGluLysGly                              145150155160                                                                  AlaArgCysThrValAlaCysMetProValProIleGluGluAlaSer                              165170175                                                                     AlaPheGlyValMetAlaValAspGluAsnAspLysIleIleGluPhe                              180185190                                                                     ValGluLysProAlaAsnProProSerMetProAsnAspProSerLys                              195200205                                                                     SerLeuAlaSerMetGlyIleTyrValPheAspAlaAspTyrLeuTyr                              210215220                                                                     GluLeuLeuGluGluAspAspArgAspGluAsnSerSerHisAspPhe                              225230235240                                                                  GlyLysAspLeuIleProLysIleThrGluAlaGlyLeuAlaTyrAla                              245250255                                                                     HisProPheProLeuSerCysValGlnSerAspProAspAlaGluPro                              260265270                                                                     TyrTrpArgAspValGlyThrLeuGluAlaTyrTrpLysAlaAsnLeu                              275280285                                                                     AspLeuAlaSerValValProGluLeuAspMetTyrAspArgAsnTrp                              290295300                                                                     ProIleArgThrTyrAsnGluSerLeuProProAlaLysPheValGln                              305310315320                                                                  AspArgSerGlySerHisGlyMetThrLeuAsnSerLeuValSerAsp                              325330335                                                                     GlyCysValIleSerGlySerValValValGlnSerValLeuPheSer                              340345350                                                                     ArgValArgValAsnSerPheCysAspIleAspSerAlaValLeuLeu                              355360365                                                                     ProGluValTrpValGlyArgSerCysArgLeuArgArgCysValIle                              370375380                                                                     AspArgAlaCysValIleProGluGlyMetValIleGlyGluAsnAla                              385390395400                                                                  GluGluAspAlaArgArgPheTyrArgSerGluGluGlyIleValLeu                              405410415                                                                     ValThrArgGluMetLeuArgLysLeuGlyHisLysGlnGluArg                                 420425430                                                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 281 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      TCTAGAAGCTTGGATATCTGGCAGCAGAAAAACAAGTAGTTGAGAACT48                            SerArgSerLeuAspIleTrpGlnGlnLysAsnLysLeuArgThr                                 151015                                                                        AAGAAGAAGAAAATGGCTTCCTCAATGATCTCCTCCCCAGCTGTTACC96                            LysLysLysLysMetAlaSerSerMetIleSerSerProAlaValThr                              202530                                                                        ACCGTCAACCGTGCCGGTGCCGGCATGGTTGCTCCATTCACCGGCCTC144                           ThrValAsnArgAlaGlyAlaGlyMetValAlaProPheThrGlyLeu                              354045                                                                        AAATCCATGGCTGGCTTCCCCACGAGGAAGACCAACAATGACATTACC192                           LysSerMetAlaGlyPheProThrArgLysThrAsnAsnAspIleThr                              505560                                                                        TCCATTGCTAGCAACGGTGGAAGAGTACAATGCATGCAGGTGTGGCCT240                           SerIleAlaSerAsnGlyGlyArgValGlnCysMetGlnValTrpPro                              657075                                                                        CCAATTGGAAAGAAGAAGTTTGAGACTCTTTCCTGGGATCC281                                  ProIleGlyLysLysLysPheGluThrLeuSerTrpAsp                                       808590                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 92 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      SerArgSerLeuAspIleTrpGlnGlnLysAsnLysLeuArgThrLys                              151015                                                                        LysLysLysMetAlaSerSerMetIleSerSerProAlaValThrThr                              202530                                                                        ValAsnArgAlaGlyAlaGlyMetValAlaProPheThrGlyLeuLys                              354045                                                                        SerMetAlaGlyPheProThrArgLysThrAsnAsnAspIleThrSer                              505560                                                                        IleAlaSerAsnGlyGlyArgValGlnCysMetGlnValTrpProPro                              65707580                                                                      IleGlyLysLysLysPheGluThrLeuSerTrpAsp                                          8590                                                                          (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 281 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      TCTAGAAGCTTGGATATCTGGCAGCAGAAAAACAAGTAGTTGAGAA46                              LeuGluAlaTrpIleSerGlySerArgLysThrSerSerGlu                                    1510                                                                          CTAAGAAGAAGAAAATGGCTTCCTCAATGATCTCCTCCCCAGCTGTTA94                            LeuArgArgArgLysTrpLeuProGlnSerProProGlnLeuLeu                                 152025                                                                        CCACCGTCAACCGTGCCGGTGCCGGCATGGTTGCTCCATTCACCGGCC142                           ProProSerThrValProValProAlaTrpLeuLeuHisSerProAla                              30354045                                                                      TCAAATCCATGGCTGGCTTCCCCACGAGGAAGACCAACAATGACATTA190                           SerAsnProTrpLeuAlaSerProArgGlyArgProThrMetThrLeu                              505560                                                                        CCTCCATTGCTAGCAACGGTGGAAGAGTACAATGCATGCAGGTGTGGC238                           ProProLeuLeuAlaThrValGluGluTyrAsnAlaCysArgCysGly                              657075                                                                        CTCCAATTGGAAAGAAGAAGTTTGAGACTCTTTCCTGGGATC280                                 LeuGlnLeuGluArgArgSerLeuArgLeuPheProGlyIle                                    808590                                                                        C281                                                                          (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 91 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      LeuGluAlaTrpIleSerGlySerArgLysThrSerSerGluLeuArg                              151015                                                                        ArgArgLysTrpLeuProGlnSerProProGlnLeuLeuProProSer                              202530                                                                        ThrValProValProAlaTrpLeuLeuHisSerProAlaSerAsnPro                              354045                                                                        TrpLeuAlaSerProArgGlyArgProThrMetThrLeuProProLeu                              505560                                                                        LeuAlaThrValGluGluTyrAsnAlaCysArgCysGlyLeuGlnLeu                              65707580                                                                      GluArgArgSerLeuArgLeuPheProGlyIle                                             8590                                                                          (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 281 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TCTAGAAGCTTGGATATCTGGCAGCAGAAAAACAAGTAGTTGAGAAC47                             LysLeuGlyTyrLeuAlaAlaGluLysGlnValValGluAsn                                    1510                                                                          TAAGAAGAAGAAAATGGCTTCCTCAATGATCTCCTCCCCAGCTGTTAC95                            GluGluGluAsnGlyPheLeuAsnAspLeuLeuProSerCysTyr                                 152025                                                                        CACCGTCAACCGTGCCGGTGCCGGCATGGTTGCTCCATTCACCGGCCT143                           HisArgGlnProCysArgCysArgHisGlyCysSerIleHisArgPro                              30354045                                                                      CAAATCCATGGCTGGCTTCCCCACGAGGAAGACCAACAATGACATTAC191                           GlnIleHisGlyTrpLeuProHisGluGluAspGlnGlnHisTyr                                 505560                                                                        CTCCATTGCTAGCAACGGTGGAAGAGTACAATGCATGCAGGTGTGGCC239                           LeuHisCysGlnArgTrpLysSerThrMetHisAlaGlyValAla                                 657075                                                                        TCCAATTGGAAAGAAGAAGTTTGAGACTCTTTCCTGGGATCC281                                 SerAsnTrpLysGluGluValAspSerPheLeuGlySer                                       8085                                                                          (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 88 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      LysLeuGlyTyrLeuAlaAlaGluLysGlnValValGluAsnGluGlu                              151015                                                                        GluAsnGlyPheLeuAsnAspLeuLeuProSerCysTyrHisArgGln                              202530                                                                        ProCysArgCysArgHisGlyCysSerIleHisArgProGlnIleHis                              354045                                                                        GlyTrpLeuProHisGluGluAspGlnGlnHisTyrLeuHisCysGln                              505560                                                                        ArgTrpLysSerThrMetHisAlaGlyValAlaSerAsnTrpLysGlu                              65707580                                                                      GluValAspSerPheLeuGlySer                                                      85                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 718 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CTCGAGATTTGTCAAATCAGGCTCAAAGATCGTTTTTCATATCGGAATGAGGATTTTATT60                TATTCTTTTAAAAATAAAGAGGTGTTGAGCTAAACAATTTCAAATCTCATCACACATATG120               GGGTCAGCCACAAAAATAAAGAACGGTTGGAACGGATCTATTATATAATACTAATAAAGA180               ATAGAAAAAGGAAAGTGAGTGAGGTGCGAGGGAGAGAATCTGTTTACTATCAGAGTCGAT240               CATGTGTCAGTTTTATCGATATGACTCTGACTTCAACTGAGTTTAAGCAATTCTGATAAG300               GCGAGGAAAATCACAGTGCTGAATCTAGAAAAATCTCATAGTGTGAGATAAGTCTCAACA360               AAAACGTTGAGTCCATAGAGGGGGTGTATGTGACACCCCAACCTCAGCAAAAGAAAACCT420               CCCCTCAAGAAGGACATTTGCGGTGCTAAACAATTTCAAGTCTCATCACACATATATATT480               ATATAATACTAATAAAGAATAGAAAAAGGAAAGGTAAACATCACTAATGACAGTTGCGGT540               GCAAAGTGAGTGAGATAATAAACATCAGTAATAGACATCACTAACTTTTATTGGTTATGT600               CAAACTCAAAATAAAATTTCTCAACTTGTTTACGTGCCTATATATACCATGCTTGTTATA660               TGCTCAAAGCACCAACAAAATTTAAAAACAATTTGAACATTTGCAAAACTAGTATGGG718                 (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 703 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      ATTTGTCAAATCAGGCTCAAAGATCGTTTTTCATATCGGAATGAGGATTTTATTTATTCT60                TTTAAAAATAAAGAGGTGTTGAGCTAAACAATTTCAAATCTCATCACACATATGGGGTCA120               GCCACAAAAATAAAGAACGGTTGGAACGGATCTATTATATAATACTAATAAAGAATAGAA180               AAAGGAAAGTGAGTGAGGTGCGAGGGAGAGAATCTGTTTACTATCAGAGTCGATCATGTG240               TCAGTTTTATCGATATGACTCTGATTTCAACTGAGTTTAAGCAATTCTGATAAGGCGAGG300               AAAATCACAGTGCTGAAATCTAGAAAAATCTCATAGTGTGAGATAAGTCTCAACAAAAAC360               GTTGAGTCCATAGAGGGGGTGTATGTGACACCCCAACCTCAGCAAAAGAAAACCTCCCCT420               CAAGAAGGACATTTGCGGTGCTAAACAATTTCAAGTCTCATCACACATATATATTATATA480               ATACTAATAAAGAATAGAAAAAGGAAAGGTAAACATCACTAATGACAGTTGCGGTGCAAA540               GTGAGTGAGATAATAAACATCAGTAATAGACATCACTAACTTTTATTGGTTATGTCAAAC600               TCAAAATAAAATTTCTCAACTTGTTTACGTGCCTATATATACCATGCTTGTTATATGCTC660               AAAGCACCAACAAAATTTAAAAACAATTTGAACATTTGCAAAA703                                (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 650 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CTCGAGATTTGTCAAATCAGGCTCAAAGATCGTTTTTCATATCGGAATGAGGATTTTATT60                TATTCTTTTAAAAATAAAGAGGTGGTGAGCTAAACAATTTCAAATCTCATCACACATATG120               GGGTCAGCCACAAAAATAAAGAACGGTTGGAACGGATCTATTATATAATACTAATAAAGA180               ATAGGAAAAGGAAAGTGAGTGAGGTGCGAGGGAGAGAATTTGTTTAATATCAGAGTCGAT240               CATGTGTCAGTTTTATCGATATGATTCTGACTTCAACTGAGTTTAAGCAATTCTGATAAG300               GCGGAGAAAATCATAGTGCTGAGTCTAGAAAAATCTCATGCAGTGTGAGATAAACCTCAA360               CAAGAACATTTGCGGTGCTAAACAATTTCAAGTCTTATCACACATATATATTATATATTA420               CTAATAAAGAATAGAAAAAGGAAAGGTAAACATCACTAATGACAGTTGCGGTGCAAAGTG480               AGTGAGATAATAAACATCACTAATAGACATCACTAACTTTTATTGGTTATGTCAAACTCA540               AAATAAAATTTCTCAACTTGTTTACGTGCCTATATATACCATGCTTGTTATATGCTCAAA600               GCACCAACAAAATTTAAAAACAATTTGAACATTTGCAAAACTAGTATGGG650                         (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 703 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      ATTTGTCAAATCAGGCTCAAAGATCGTTTTTCATATCGGAATGAGGATTTTATTTATTCT60                TTTAAAAATAAAGAGGTGTTGAGCTAAACAATTTCAAATCTCATCACACATATGGGGTCA120               GCCACAAAAATAAAGAACGGTTGGAACGGATCTATTATATAATACTAATAAAGAATAGAA180               AAAGGAAAGTGAGTGAGGTGCGAGGGAGAGAATCTGTTTACTATCAGAGTCGATCATGTG240               TCAGTTTTATCGATATGACTCTGATTTCAACTGAGTTTAAGCAATTCTGATAAGGCGAGG300               AAAATCACAGTGCTGAAATCTAGAAAAATCTCATAGTGTGAGATAAGTCTCAACAAAAAC360               GTTGAGTCCATAGAGGGGGTGTATGTGACACCCCAACCTCAGCAAAAGAAAACCTCCCCT420               CAAGAAGGACATTTGCGGTGCTAAACAATTTCAAGTCTCATCACACATATATATTATATA480               ATACTAATAAAGAATAGAAAAAGGAAAGGTAAACATCACTAATGACAGTTGCGGTGCAAA540               GTGAGTGAGATAATAAACATCAGTAATAGACATCACTAACTTTTATTGGTTATGTCAAAC600               TCAAAATAAAATTTCTCAACTTGTTTACGTGCCTATATATACCATGCTTGTTATATGCTC660               AAAGCACCAACAAAATTTAAAAACAATTTGAACATTTGCAAAA703                                (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2000 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GGATCCATTAGGACTAGATAATGAAAAGAAACCGTTTTTTTAATACCTCGGCTGCTATTG60                CCATTTCGATTGCATTAAATACTTTTTTTTGTAGCATGCAGACGATTGCTGCTGAACCAG120               AAGAAACTTATCTTGATTTTCGTAAGGAGACGATATATTTTCTATTCCTTGATCGTTTCA180               GCGATGGAGATCCAAGTAATAATGCAGGGTTTAATTCTGCAACCTACGATCCTAATAATT240               TAAAAAAATATACTGGAGGAGATCTCCGGGGGTTGATTAATAAACTACCCTATTTAAAAT300               CACTTGGTGTTACTTCAATCTGGATTACTCCCCCAATCGATAATGTGAATAATACTGATG360               CTGCTGGCAATACTGGATATCATGGTTATTGGGGAAGAGATTATTTTCGTATAGATGAAC420               ATTTTGGCAATCTCGATGATTTCAAAGAACTGACTAGTTTGATGCATAGTCCTGATTATA480               ATATGAAACTGGTTCTTGATTATGCCCCTAATCATTCGAATGCTAATGATGAAAATGAAT540               TTGGTGCACTATATCGTGATGGTGTGTTTATTACTGATTATCCTACAGATGTTGCCGCCA600               ATACGGGCTGGTATCATCACAATGGTGGGGTAACGAACTGGAATGATTTCTTCCAAGTGA660               AGAATCATAATCTATTCAATCTATCAGACCTCAATCAATCCAATACTGATGTCTACCAGT720               ACTTGTTGGATGGCTCTAAATTTTGGATCGATGCTGGTGTGGATGCTATCAGGATTGATG780               CCATCAAGCATATGGACAAGTCTTTTATACAGAAATGGACCAGCGATATTTATGATTACA840               GTAAGTCTATCGGCCGGGAAGGATTTTTTTTCTTCGGTGAATGGTTTGGTGCCAGTGCGA900               ATACTACAACAGGTGTTGATGGTAATGCTATCGATTACGCCAACACTTCCGGGTCAGCGT960               TGCTGGATTTTGGATTCCGCGATACTTTAGAAAGAGTTTTGGTAGGACGTAGCGGAAATA1020              CAATGAAAACGTTAAATAGTTATCTGATAAAAAGACAAACAGTCTTTACCAGTGATGACT1080              GGCAGGTTGTTTTTATGGATAACCATGATATGGCACGCATTGGTACCGCTCTGCGTTCAA1140              ACGCCACTACTTTTGGTCCTGGAAATAATGAAACCGGTGGAAGTCAGAGTGAAGCTTTTG1200              CTCAGAAACGTATAGACCTCGGTCTGGTTGCGACAATGACTGTACGTGGTATTCCTGCCA1260              TTTATTATGGTACTGAACATTATGCCGCTAACTTTACCTCTAACAGTTTTGGTCAAGTTG1320              GCAGTGATCCTTACAACCGAGAGAAAATGCCAGGATTTGATACGGAAAGTGAGGCTTTCT1380              CCATTATTAAAACACTGGGTGACCTAAGGAAAAGTAGCCCGGCAATTCAAAATGGAACTT1440              ATACTGAACTATGGGTTAATGATGATATATTAGTATTTGAGCGGCGTTCTGGGAACGATA1500              TTGTTATTGTTGCACTTAATCGTGGTGAGGCTAACACAATTAATGTTAAAAATATAGCGG1560              TTCCTAATGGGGTATATCCGAGTTTGATTGGGAATAATAGTGTTTCAGTAGCAAATAAAC1620              AGGCAACACTAACACTTATGCAAAATGAAGCTGTTGTCATTCGCTCACAATCAGATGATG1680              CGGAGAACCCTACAGTACAAAGCATAAACTTCGCATGTAATAACGGTTATACGATTTCAG1740              GTCAAAGTGTTTATATTATTGGTAATATACCTCAGTTAGGTGGTTGGGACTTAACTAAAG1800              CGGTAAAAATATCACCGACACAATATCCACAATGGAGTGCGAGCTTAGAGCTTCCTTCTG1860              ACTTAAATGTTGAATGGAAGTGTGTGAAACGTAATGAAACCAATCCGACGGCTAATGTTG1920              AGTGGCAGTCTGGTGCAAATAACCAGTTCAATAGCAATGACACACAAACAACGAATGGCT1980              CGTTTTAATTAAAAGTCGAC2000                                                      (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1988 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: NO.                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      ATTAGGACTAGATAATGAAAAGAAACCGTTTTTTTAATACCTCGGCTGCTATTGCCATTT60                CGATTGCATTAAATACTTTTTTTTGTAGCATGCAGACGATTGCTGCTGAACCAGAAGAAA120               CTTATCTTGATTTTCGTAAGGAGACGATATATTTTCTATTCCTTGATCGTTTCAGCGATG180               GAGATCCAAGTAATAATGCAGGGTTTAATTCTGCAACCTACGATCCTAATAATTTAAAAA240               AATATACTGGAGGAGATCTCCGGGGGTTGATTAATAAACTACCCTATTTAAAATCACTTG300               GTGTTACTTCAATCTGGATTACTCCCCCAATCGATAATGTGAATAATACTGATGCTGCTG360               GCAATACTGGATATCATGGTTATTGGGGAAGAGATTATTTTCGTATAGATGAACATTTTG420               GCAATCTCGATGATTTCAAAGAACTGACTAGTTTGATGCATAGTCCTGATTATAATATGA480               AACTGGTTCTTGATTATGCCCCTAATCATTCGAATGCTAATGATGAAAATGAATTTGGTG540               CACTATATCGTGATGGTGTGTTTATTACTGATTATCCTACGAATGTTGCCGCCAATACGG600               GCTGGTATCATCACAATGGTGGGGTAACGAACTGGAATGATTTCTTCCAAGTGAAGAATC660               ATAATCTATTCAATCTATCAGACCTCAATCAATCCAATACTGATGTCTACCAGTACTTGT720               TGGATGGTTCTAAATTTTGGATCGATGCTGGTGTGGATGCTATCAGGATTGATGCCATCA780               AGCATATGGACAAGTCTTTTATACAGAAATGGACCAGCGATATTTATGATTACAGTAAGT840               CTATCGGCCGGGAAGGATTTTTTTTCTTCGGTGAATGGTTTGGTGCCAGTGCGAATACTA900               CAACAGGTGTTGATGGTAATGCTATCGATTACGCCAACACTTCCGGGTCAGCGTTGCTGG960               ATTTTGGATTCCGCGATACTTTAGAAAGAGTTTTGGTAGGACGTAGCGGAAATACAATGA1020              AAACGTTAAATAGTTATCTGATAAAAAGACAAACAGTCTTTACCAGTGATGACTGGCAGG1080              TTGTTTTTATGGATAACCATGATATGGCACGCATTGGTACCGCTCTGCGTTCAAACGCCA1140              CTACTTTTGGTCCTGGAAATAATGAAACCGGTGGAAGTCAGAGTGAAGCTTTTGCTCAGA1200              AACGTATAGACCTCGGTCTGGTTGCGACAATGACTGTACGTGGTATTCCTGCCATTTATT1260              ATGGTACTGAACATTATGCCGCTAACTTTACCTCTAACAGTTTTGGTCAAGTTGGCAGTG1320              ATCCTTACAACCGAGAGAAAATGCCAGGATTTGATACGGAAAGTGAGGCTTTCTCCATTA1380              TTAAAACACTGGGTGACCTAAGGAAAAGTAGCCCGGCAATTCAAAATGGAACTTATACTG1440              AACTATGGGTTAATGATGATATATTAGTATTTGAGCGGCGTTCTGGGAACGATATTGTTA1500              TTGTTGCACTTAATCGTGGTGAGGCTAACACAATTAATGTTAAAAATATAGCGGTTCCTA1560              ATGGGGTATATCCGAGTTTGATTGGGAATAATAGTGTTTCAGTAGCAAATAAACGGACAA1620              CACTAACACTTATGCAAAATGAAGCTGTTGTCATTCGCTCACAATCAGATGATGCGGAGA1680              ACCCTACAGTACAAAGCATAAACTTCACATGTAATAACGGTTATACGATTTCAGGTCAAA1740              GTGTTTATATTATTGGTAATATACCTCAGTTAGGTGGTTGGGACTTAACTAAAGCGGTAA1800              AAATATCACCGACACAATATCCACAATGGAGTGCGAGCTTAGAGCTTCCTTCTGACTTAA1860              ATGTTGAATGGAAGTGTGTGAAACGTAATGAAACCAATCCGACGGCTAATGTTGAGTGGC1920              AGTCTGGTGCAAATAACCAGTTCAATAGCAATGACACACAAACAACGAATGGCTCGTTTT1980              AATTAAAA1988                                                                  (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 655 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      MetLysArgAsnArgPhePheAsnThrSerAlaAlaIleAlaIleSer                              151015                                                                        IleAlaLeuAsnThrPhePheCysSerMetGlnThrIleAlaAlaGlu                              202530                                                                        ProGluGluThrTyrLeuAspPheArgLysGluThrIleTyrPheLeu                              354045                                                                        PheLeuAspArgPheSerAspGlyAspProSerAsnAsnAlaGlyPhe                              505560                                                                        AsnSerAlaThrTyrAspProAsnAsnLeuLysLysTyrThrGlyGly                              65707580                                                                      AspLeuArgGlyLeuIleAsnLysLeuProTyrLeuLysSerLeuGly                              859095                                                                        ValThrSerIleTrpIleThrProProIleAspAsnValAsnAsnThr                              100105110                                                                     AspAlaAlaGlyAsnThrGlyTyrHisGlyTyrTrpGlyArgAspTyr                              115120125                                                                     PheArgIleAspGluHisPheGlyAsnLeuAspAspPheLysGluLeu                              130135140                                                                     ThrSerLeuMetHisSerProAspTyrAsnMetLysLeuValLeuAsp                              145150155160                                                                  TyrAlaProAsnHisSerAsnAlaAsnAspGluAsnGluPheGlyAla                              165170175                                                                     LeuTyrArgAspGlyValPheIleThrAspTyrProThrAspValAla                              180185190                                                                     AlaAsnThrGlyTrpTyrHisHisAsnGlyGlyValThrAsnTrpAsn                              195200205                                                                     AspPhePheGlnValLysAsnHisAsnLeuPheAsnLeuSerAspLeu                              210215220                                                                     AsnGlnSerAsnThrAspValTyrGlnTyrLeuLeuAspGlySerLys                              225230235240                                                                  PheTrpIleAspAlaGlyValAspAlaIleArgIleAspAlaIleLys                              245250255                                                                     HisMetAspLysSerPheIleGlnLysTrpThrSerAspIleTyrAsp                              260265270                                                                     TyrSerLysSerIleGlyArgGluGlyPhePhePhePheGlyGluTrp                              275280285                                                                     PheGlyAlaSerAlaAsnThrThrThrGlyValAspGlyAsnAlaIle                              290295300                                                                     AspTyrAlaAsnThrSerGlySerAlaLeuLeuAspPheGlyPheArg                              305310315320                                                                  AspThrLeuGluArgValLeuValGlyArgSerGlyAsnThrMetLys                              325330335                                                                     ThrLeuAsnSerTyrLeuIleLysArgGlnThrValPheThrSerAsp                              340345350                                                                     AspTrpGlnValValPheMetAspAsnHisAspMetAlaArgIleGly                              355360365                                                                     ThrAlaLeuArgSerAsnAlaThrThrPheGlyProGlyAsnAsnGlu                              370375380                                                                     ThrGlyGlySerGlnSerGluAlaPheAlaGlnLysArgIleAspLeu                              385390395400                                                                  GlyLeuValAlaThrMetThrValArgGlyIleProAlaIleTyrTyr                              405410415                                                                     GlyThrGluHisTyrAlaAlaAsnPheThrSerAsnSerPheGlyGln                              420425430                                                                     ValGlySerAspProTyrAsnArgGluLysMetProGlyPheAspThr                              435440445                                                                     GluSerGluAlaPheSerIleIleLysThrLeuGlyAspLeuArgLys                              450455460                                                                     SerSerProAlaIleGlnAsnGlyThrTyrThrGluLeuTrpValAsn                              465470475480                                                                  AspAspIleLeuValPheGluArgArgSerGlyAsnAspIleValIle                              485490495                                                                     ValAlaLeuAsnArgGlyGluAlaAsnThrIleAsnValLysAsnIle                              500505510                                                                     AlaValProAsnGlyValTyrProSerLeuIleGlyAsnAsnSerVal                              515520525                                                                     SerValAlaAsnLysGlnAlaThrLeuThrLeuMetGlnAsnGluAla                              530535540                                                                     ValValIleArgSerGlnSerAspAspAlaGluAsnProThrValGln                              545550555560                                                                  SerIleAsnPheAlaCysAsnAsnGlyTyrThrIleSerGlyGlnSer                              565570575                                                                     ValTyrIleIleGlyAsnIleProGlnLeuGlyGlyTrpAspLeuThr                              580585590                                                                     LysAlaValLysIleSerProThrGlnTyrProGlnTrpSerAlaSer                              595600605                                                                     LeuGluLeuProSerAspLeuAsnValGluTrpLysCysValLysArg                              610615620                                                                     AsnGluThrAsnProThrAlaAsnValGluTrpGlnSerGlyAlaAsn                              625630635640                                                                  AsnGlnPheAsnSerAsnAspThrGlnThrThrAsnGlySerPhe                                 645650655                                                                     (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 655 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      MetLysArgAsnArgPhePheAsnThrSerAlaAlaIleAlaIleSer                              151015                                                                        IleAlaLeuAsnThrPhePheCysSerMetGlnThrIleAlaAlaGlu                              202530                                                                        ProGluGluThrTyrLeuAspPheArgLysGluThrIleTyrPheLeu                              354045                                                                        PheLeuAspArgPheSerAspGlyAspProSerAsnAsnAlaGlyPhe                              505560                                                                        AsnSerAlaThrTyrAspProAsnAsnLeuLysLysTyrThrGlyGly                              65707580                                                                      AspLeuArgGlyLeuIleAsnLysLeuProTyrLeuLysSerLeuGly                              859095                                                                        ValThrSerIleTrpIleThrProProIleAspAsnValAsnAsnThr                              100105110                                                                     AspAlaAlaGlyAsnThrGlyTyrHisGlyTyrTrpGlyArgAspTyr                              115120125                                                                     PheArgIleAspGluHisPheGlyAsnLeuAspAspPheLysGluLeu                              130135140                                                                     ThrSerLeuMetHisSerProAspTyrAsnMetLysLeuValLeuAsp                              145150155160                                                                  TyrAlaProAsnHisSerAsnAlaAsnAspGluAsnGluPheGlyAla                              165170175                                                                     LeuTyrArgAspGlyValPheIleThrAspTyrProThrAsnValAla                              180185190                                                                     AlaAsnThrGlyTrpTyrHisHisAsnGlyGlyValThrAsnTrpAsn                              195200205                                                                     AspPhePheGlnValLysAsnHisAsnLeuPheAsnLeuSerAspLeu                              210215220                                                                     AsnGlnSerAsnThrAspValTyrGlnTyrLeuLeuAspGlySerLys                              225230235240                                                                  PheTrpIleAspAlaGlyValAspAlaIleArgIleAspAlaIleLys                              245250255                                                                     HisMetAspLysSerPheIleGlnLysTrpThrSerAspIleTyrAsp                              260265270                                                                     TyrSerLysSerIleGlyArgGluGlyPhePhePhePheGlyGluTrp                              275280285                                                                     PheGlyAlaSerAlaAsnThrThrThrGlyValAspGlyAsnAlaIle                              290295300                                                                     AspTyrAlaAsnThrSerGlySerAlaLeuLeuAspPheGlyPheArg                              305310315320                                                                  AspThrLeuGluArgValLeuValGlyArgSerGlyAsnThrMetLys                              325330335                                                                     ThrLeuAsnSerTyrLeuIleLysArgGlnThrValPheThrSerAsp                              340345350                                                                     AspTrpGlnValValPheMetAspAsnHisAspMetAlaArgIleGly                              355360365                                                                     ThrAlaLeuArgSerAsnAlaThrThrPheGlyProGlyAsnAsnGlu                              370375380                                                                     ThrGlyGlySerGlnSerGluAlaPheAlaGlnLysArgIleAspLeu                              385390395400                                                                  GlyLeuValAlaThrMetThrValArgGlyIleProAlaIleTyrTyr                              405410415                                                                     GlyThrGluHisTyrAlaAlaAsnPheThrSerAsnSerPheGlyGln                              420425430                                                                     ValGlySerAspProTyrAsnArgGluLysMetProGlyPheAspThr                              435440445                                                                     GluSerGluAlaPheSerIleIleLysThrLeuGlyAspLeuArgLys                              450455460                                                                     SerSerProAlaIleGlnAsnGlyThrTyrThrGluLeuTrpValAsn                              465470475480                                                                  AspAspIleLeuValPheGluArgArgSerGlyAsnAspIleValIle                              485490495                                                                     ValAlaLeuAsnArgGlyGluAlaAsnThrIleAsnValLysAsnIle                              500505510                                                                     AlaValProAsnGlyValTyrProSerLeuIleGlyAsnAsnSerVal                              515520525                                                                     SerValAlaAsnLysArgThrThrLeuThrLeuMetGlnAsnGluAla                              530535540                                                                     ValValIleArgSerGlnSerAspAspAlaGluAsnProThrValGln                              545550555560                                                                  SerIleAsnPheThrCysAsnAsnGlyTyrThrIleSerGlyGlnSer                              565570575                                                                     ValTyrIleIleGlyAsnIleProGlnLeuGlyGlyTrpAspLeuThr                              580585590                                                                     LysAlaValLysIleSerProThrGlnTyrProGlnTrpSerAlaSer                              595600605                                                                     LeuGluLeuProSerAspLeuAsnValGluTrpLysCysValLysArg                              610615620                                                                     AsnGluThrAsnProThrAlaAsnValGluTrpGlnSerGlyAlaAsn                              625630635640                                                                  AsnGlnPheAsnSerAsnAspThrGlnThrThrAsnGlySerPhe                                 645650655                                                                     __________________________________________________________________________

We claim:
 1. A nucleic acid construct comprising a chimeric gene whichis expressed in plant cells said chimeric gene comprising a nucleotidesequence encoding a cyclodextrin glycosyltransferase enzyme operablylinked with a promoter which is operable in a plant cell.
 2. Theconstruct of claim 1 wherein said nucleotide sequence isdeoxyribonucleic acid.
 3. The construct of claim 1 having a sequencecomprising, in the 5'-3' direction of transcription, a transcriptioninitiation region, a translational initiation region, an open readingframe sequence encoding said enzyme, and a transcription/translationaltermination region, wherein said regulatory regions are capable offunctioning in a plant cell.
 4. The construct of claim 3 wherein saidtranscription and translational initiation regions comprise at least aportion of the regulatory region-5' to a patatin gene from Solanumtuberosum.
 5. The construct of claim 4, wherein said transcription andtranslational initiation regions are from Solanum tuberosum var.Kennebec.
 6. The construct of claim 4, wherein said transcription andtranslational initiation regions are from Solanum tuberosum var. RussetBurbank.
 7. The construct of claim 4, wherein said transcription andtranslational initiation regions are from a gene which is preferentiallyexpressed in a potato tuber.
 8. The construct of claim 3 furthercomprising a T-DNA border.
 9. The construct of claim 1, wherein saidcyclodextrin glycosyltransferase enzyme is a Klebsiella pneumoneaeenzyme.
 10. The construct of claim 1, further comprising a sequencecoding for a marker capable of being identified and selected in aeukaryotic cell containing said sequence.
 11. The construct of claim 3further comprising: a nucleic acid sequence, bound to the 3'-end of saidtranscription and translational initiation region, and encoding atransit peptide joined in reading frame at the 5'-terminus of said openreading frame sequence, where the transit peptide is capable ofdirecting transport of the expression product of said enzyme-encodingsequence, to at least one discrete location in a plant.
 12. Theconstruct of claim 11, wherein said transcription initiation region isfrom a gene preferentially expressed in starch-containing tissue of saidplant cell.
 13. The construct of claim 12, wherein said gene is apatatin gene derived from Solanum tuberosum.
 14. The construct of claim13, wherein said patatin gene is derived from a member selected from thegroup consisting of Solanum tuberosum var. Kennebac and Solanumtuberosum var. Russett Burbank.
 15. A method to modify a starch storageorgan comprising growing a plant host under conditions which permit theformation of a starch storage organ, having in its genome a nucleic acidconstruct which is capable of expressing a chimeric gene comprising anucleotide sequence encoding a cyclodextrin zlycosvltransferase enzymeoperably linked with a promoter, preferentially in said starch storageorgan.
 16. The method of claim 15 wherein the plant host is selectedfrom the group consisting of Zea species Triticum species, Secalespecies, Sorghum species, Oryza species, Solanum species, Ipomoeaspecies, Discorea species, Manihot species, Marantaceae species,Cycadaceae species, Cannaceae species, Zingiberaceae species, Palmaespecies and Cycadales species.
 17. A method for producing reservepolysaccharide degradation products in plants which comprises:a) stablymodifying at least one plant host cell with a nucleic acid constructcomprising, in the 5'-3' direction of transcription:i) a transcriptionaland translational initiation region functional in a plant host cell; andii) a structural gene coding for a cyclodextrin gvcosvltransferaseenzyme gene sequence, said sequence being under the transcriptionalcontrol of said plant host; and b) maintaining the plant host containingsaid construct under conditions which permit the expression of afunctional cyclodextrin lycosultransferase enzyme encoded by said enzymegene sequence.
 18. The method of claim 17, wherein said transcriptionaland translational initiation region comprises at least a portion of aregion 5' to a patatin gene from Solanum tuberosum.
 19. The method ofclaim 18, wherein said transcriptional and translational initiationregion is from Solanum tuberosum var. Kennebec.
 20. The method of claim18, wherein said transcriptional and translational initiation region isfrom Solanum tuberosum var. Russet Burbank.
 21. The method of claim 17,wherein said cyclodextrin glycosyltransferase enzyme is a Klebsiellapneumoneae enzyme.
 22. The method of claim 17 wherein said nucleic acidconstruct further comprises a sequence encoding a transit peptide inreading frame at the 5'-terminus of said enzyme coding sequence, wherethe transit peptide is capable of directing transport of the expressionproduct of said enzyme encoding sequence to at least one discretelocation in the plant host organism.
 23. The method of claim 17 whereinthe plant host is selected from the group consisting of Zea species,Triticum species, Secale species, Sorghum species, Oryza species Solanumspecies, Ipomoea species, Discorea species, Manihot species, Marantaceaespecies, Cycadaceae species, Cannaceae species, Zingiberaceae species,Palmae species and Cycadales species.
 24. A plant starch storage organhaving a modified specific gravity produced by the method of claim 15.25. A plant starch storage organ produced by the method of claim
 15. 26.A plant host cell comprising the nucleic acid construct of claim 1wherein said cell is capable of expressing at least one functionalcyclodextrin glycosultransferase enzyme.
 27. The plant host cell ofclaim 26, wherein said cyclodextrin glycosyltransferase enzyme is aKlevsiella pneumoneae enzyme.
 28. The plant host cell of claim 26wherein the plant host is selected from the group consisting of Zeaspecies, Triticum species, Secale species, Sorghum species, Oryzaspecies, Solanum species Ipomoea species, Discorea species, Manihotspecies, Marantaceae species, Cycadaceae species, Cannaceae species,Zingiberaceae species, Palmae species and Cycadales species.
 29. Atransformed plant comprising the nucleic acid construct of claim 1wherein said plant is capable of producing at least one cyclodextrin asa starch degradation product.
 30. The plant of claim 29 wherein saidcyclodextrin comprises at least one member selected from the groupconsisting of α-cyclodextrins, β-cyclodextrins and γ-cyclodextrins. 31.The plant of claim 30 wherein the plant is selected from the groupconsisting of Zea species, Triticum species, Secale species, Sorghumspecies, Oryza species, Solanum species, Ipomoea species Discoreaspecies, Manihot species, Marantaceae species, Cycadaceae species,Cannaceae species, Zingiberaceae species, Palmae species and Cycadalesspecies.
 32. The construct of claim 9, wherein said nucleotide sequenceis a plant preferred coding sequence.
 33. The method of claim 21,wherein said nucleotide sequence is a plant-preferred coding sequence.34. The plant host cell of claim 27, wherein said nucleotide sequence isa plant-preferred coding sequence.
 35. The method of claim 16 whereinthe plant host is selected from the group consisting of Zea mays, Secalecereale, Sorghum bicolor, Oryza sativa, Solanum tuberosum, Ipomoeabatatas, and Manihot esculenta.
 36. The method of claim 15, wherein theplant host is a Triticum aestivum×Secale cereale hybrid.
 37. The methodof claim 23 wherein the plant host is selected from the group consistingof Zea mays, Secale cereale, Sorghum bicolor, Oryza sativa, Solanumtuberosum, Ipomoea batatas, and Manihot esculenta.
 38. The method ofclaim 17, wherein the plant host is a Triticum aestivum×Secale cerealehybrid.
 39. The plant host cell of claim 28, wherein the plant host isselected from the group consisting of Zea mays, Secale cereale, Sorghumbicolor, Oryza sativa, Solanum tuberosum, Ipomoea batatas, and Manihotesculenta.
 40. The plant host cell of claim 26, wherein the plant hostis a Triticum aestivum×Secale cereale hybrid.
 41. The plant of claimwherein the plant is selected from the group consisting of Zea mays,Secale cereale, Sorghum bicolor, Oryza sativa, Solanum tuberosum,Ipomoea batatas, and Manihot esculenta.
 42. The plant of claim 30,wherein the plant is a Triticum aestivum×Secale cereale hybrid.